CN1930288B - Cells of which genome is modified - Google Patents

Cells of which genome is modified Download PDF

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CN1930288B
CN1930288B CN038133997A CN03813399A CN1930288B CN 1930288 B CN1930288 B CN 1930288B CN 038133997 A CN038133997 A CN 038133997A CN 03813399 A CN03813399 A CN 03813399A CN 1930288 B CN1930288 B CN 1930288B
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antibody
cell
sugar chain
gene
fucose
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CN1930288A (en
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大贯尚子
佐藤光男
森胜弘
山野和也
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Kyowa Kirin Co Ltd
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Kyowa Hakko Kirin Co Ltd
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Abstract

A cell in which genome is modified so as to have a more decreased or deleted activity of an enzyme relating to modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through alpha-bond in a complex N-glycoside-linked sugar chain than its parent cell, and a process for producing an antibody composition using the cell.

Description

The adorned cell of genome
Technical field
The present invention relates to the adorned cell of a kind of genome; Compare with its parental cell; Have reduction or removed the activity with sugar chain modified involved enzyme; The 6-position that the 1-position of Fucose is connected the N-acetylglucosamine of sugar chain complex body reduction end in the said sugar chain with the N-glucosides combines through α-key, and adopts this cell to produce the process of antibody molecule.
Background technology
In the Fc district of IgG antibody-like, exist two N-glucosides to connect the sugar chain binding site.In serum IgG, on the sugar chain binding site, combined one to have a plurality of ramose mixture type sugar chains usually, sialyl that wherein adds or five equilibrium N-acetylglucosamine are seldom.Known be added on the non-reduced end of mixture type sugar chain with regard to semi-lactosi be added into Fucose certain variety arranged on the N-acetylglucosamine of reduction end [Biochemistry (biological chemistry), 36, 130 (1997)].
This sugar chain structure thought and can confirm through the sugar chain gene, just the gene of the glycolytic ferment of the gene of the glycosyltransferase of synthetic sugar chain and hydrolysis sugar chain.
The N-glucosides connects the synthetic of sugar chain and is described below.
Gp is modified with sugar chain in endoplasmic reticulum (being called " ER " later on) chamber.Connect in the biosynthesizing step of sugar chain at the N-glucosides, big relatively sugar chain is transferred on the polypeptied chain that in the ER chamber, extends.In transition process, sugar chain joins on the phosphate group of the long-chain lipid carrier of being made up of about 20 α-isoprene units at first continuously, is called phosphoric acid dolichol (after this being sometimes referred to as " P-Dol ").That is, the N-acetylglucosamine is transferred to the phosphoric acid dolichol and forms GlcNAc-P-P-Dol, shifts another GlcNAc then, thereby forms GlcNAc-GlcNAc-P-P-Dol.Next step, 5 seminoses (after this seminose is called as " Man " sometimes) are transferred, and therefore form (Man) 5-(GlcNAc) 2-P-P-Dol, four Man and three glucose (after this glucose is called as " Glc " sometimes) are transferred then.Therefore, formed the sugar chain precursor, (Glc) 3-(Man) 9-(GlcNAc) 2-P-P-Dol is called and has formed core oligosaccharide.The sugar chain precursor that contains 14 sugar is used as a group and is transferred to and contains in the endoplasmic on l-asparagine-X-Serine or the l-asparagine-X-Threonine polypeptide of sequence.In reaction, the phosphoric acid dolichol (P-P-Dol) that is bonded on the core oligosaccharide is released, but becomes the phosphoric acid dolichol once more through the hydrolytic action of Pyrophosphate phosphohydrolase, and circulates again.In sugar chain and the processing that begins sugar chain after polypeptide combines immediately.That is, three Glc are deleted on endoplasmic reticulum with one or two Man, known α-1, and 2-glucuroide I, α-1,3-glucuroide II and α-1, and the 2-mannosidase is relevant with the deletion effect.Be transferred on the golgi body at gp processed on the endoplasmic reticulum, carried out different modifications.The part in the pond of golgi body (cis); There is the relevant N-acetylglucosamine phosphotransferase that adds seminose phosphoric acid; N-acetylglucosamine 1-phosphodiester alpha-N-acetamino glucuroide and alpha-Mannosidase I, and the Man residue is reduced to 5.Middle portion at golgi body; Existence relates to the N-acetylglucosaminyl transferase I (GnTI) in the interpolation of first GlcNAc of the outside of mixture type N-glucosides connection sugar chain; Relate to the alpha-Mannosidase II that deletes two Man; Relate to from the outside and add the N-acetylglucosaminyl transferase II (GnTII) of second GlcNAc and relate to the α 1 on the N-acetylglucosamine that Fucose is added into reduction end, 6-fucosyltransferase.In the trans of golgi body part, there are galactotransferase relevant and sialytransferase or the analogue relevant with the interpolation of sialyl such as N-n acetylneuraminic acid n with the interpolation of semi-lactosi.It is that effect through these different enzymes forms that known N-glucosides connects sugar chain.
With regard to the sugar chain in the antibody, it is reported that interpolation that Fucose connects from sugar chain reduction end N-acetylglucosamine to antibody N-glucosides modifies cell-mediated cytotoxic activity (after this being called " ADCC is active ") that the antibody that greatly changed antibody relies on (WO00/61739).This report shows that the structure of sugar chain plays a significant role in the effector functions of people's IgG antibody 1 hypotype.
Usually, consider to prepare through using genetic recombination techniques as most of humanized antibodies of medicine, use zooblast, the Chinese hamster ovary celI tissue-derived like Chinese hamster ovary produces as host cell.But as stated; Because sugar chain structure is brought into play important effect in the effector functions of antibody; And in the sugar chain structure of the gp of host cell expression, observe some differences, need exploitation to be used to produce the host cell of antibody with higher effector functions.
Also carried out modifying trial through the sugar chain structure that imports the gp that produces about sugar chain modified enzyme gene; As its example; Existing report 1) possibly produce a kind of albumen; Wherein a large amount of sialyls are through with rat beta galactose glycosides-α-2, and the 6-sialytransferase imports in the Chinese hamster ovary celI and is added into [J.Biol.Chem. (journal of biological chemistry) on the non-reduced end of sugar chain 261,13848 (1989)]; 2) through people's beta galactose glycosides-2-α-fucosyltransferase is transferred in the mouse Lcell; Possibly express a kind of H antigen, wherein Fucose (after this being called " Fuc ") is added into (Fuc α 1-2Gal β 1-) [Science (science) on the non-reduced end of sugar chain 252,1668 (1991)]; With 3) through using a kind of β-1 that imported; The Chinese hamster ovary celI of 4-N-acetylglucosaminyl transferase III (GnTIII) produces a kind of antibody; Thereby possibly produce the very high antibody [Glycobiology (glycobiology) of five equilibrium N-acetylglucosamine adding proportion that a kind of N-glucosides connects sugar chain 5, 813 (1995): WO 99/54342].When having imported the expressing cho cell antibody of GnTIII through use, said antibody shows that the ADCC high 16 times than the antibody of expressing in the parental cell is active.Yet because existing report GnTIII or β-1, the over-expresses of 4-N-acetylglucosaminyl transferase V (GnTV) demonstrates the toxicity to Chinese hamster ovary celI, so it is not suitable for producing antibody drug.
To the also existing report of the production example of gp; The sugar chain structure that wherein produces can change as host cell through using a kind of two mutants; Activity about sugar chain modified enzyme gene in this two mutants is changed; As its example; Existing report uses a kind of mutant clone of Chinese hamster ovary celI to produce to have high mannose type the antibody of (high mannose type) sugar chain structure, and 4-N-acetylglucosaminyl transferase I (GnTI) is deleted [J.Immunol. (Journal of Immunology) in this mutant clone 160,3393 (1998)].In addition; Reported a kind of antibody expression, it has a kind of sugar chain structure, and wherein sialyl is not added into the non-reduction end side of sugar chain; With through using cmp sialic acid transhipment or UDP-semi-lactosi to transport the antibody expression example that does not add semi-lactosi that sub-defective clone produces; But do not find to have the enhanced effector functions, be suitable as the antibody [J.Immunol. (Journal of Immunology) of medicine 160,3393 (1998)].Because the mutant clone that has obtained is to handle through mutagenic compound to import the clone that random mutation obtains, they are not suitable for as the clone who is used to produce pharmaceutical formulations.
Therefore, for the sugar chain structure of the gp of modifying preparation, many trials of the relevant sugar chain modified enzymic activity of control in host cell have been carried out.Because sugar chain modified mechanism is variation and complicated, the physiological action of talkative sugar chain is not illustrated fully, so present case is to want TE and mistake but in fact.The effector functions of specifically little by little having illustrated antibody receives the influence of sugar chain structure very big, but can produce the host cell also not acquisition at present of the antibody molecule with only sugar chain structure modification.
Summary of the invention
The present invention relates to following (1) to (43).
(1) a kind of modifying factor group is so that cell lower with its parental cell of specific activity of sugar chain modified involved enzyme or that disappear, in the said sugar chain 1 of Fucose with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end pass through α-key connection.
(2) according to the cell of (1), the genomic gene of the sugar chain modified relevant enzyme of wherein encoding is knocked out, in the said sugar chain 1 of Fucose with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end pass through α-key connection.
(3) cell of basis (1) or (2); All allelotrope on the genome of the sugar chain modified relevant enzyme of encoding are knocked out, said sugar chain modified in 1 of Fucose with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end pass through α-key connection.
(4) according to (1) to (3) cell in each; With sugar chain modified involved enzyme be α 1; The 6-fucosyltransferase, in the said sugar chain 1 of Fucose with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end connect through α-key.
(5) according to the cell of (4), α 1, and the 6-fucosyltransferase is to be selected from following (a) protein to the dna encoding of (d):
(a) contain the DNA of the nucleotide sequence shown in the SEQ ID NO:1;
(b) contain the DNA of the nucleotide sequence shown in the SEQ ID NO:2;
(c) under stringent condition,, and has α 1, the active DNA of 6-fucosyltransferase with the DNA hybridization that comprises the nucleotide sequence shown in the SEQ ID NO:1;
(d) under stringent condition,, and has α 1, the active DNA of 6-fucosyltransferase with the DNA hybridization that comprises the nucleotide sequence shown in the SEQ ID NO:2.
(6) according to the cell of (4), α 1, and the 6-fucosyltransferase is to be selected from following (a) and (b), (c), (d), (e) and protein (f):
(a) contain the protein of aminoacid sequence shown in the SEQ ID NO:4;
(b) contain the protein of aminoacid sequence shown in the SEQ ID NO:5;
(c) contain the aminoacid sequence that at least one amino acid is deleted, replaces, inserted and/or adds in the aminoacid sequence shown in the SEQ ID NO:4, and have α 1, the active protein of 6-fucosyltransferase;
(d) contain the aminoacid sequence that at least one amino acid is deleted, replaces, inserted and/or adds in the aminoacid sequence shown in the SEQ ID NO:5, and have α 1, the active protein of 6-fucosyltransferase;
(e) contain with aminoacid sequence shown in the SEQ ID NO:4 and have 80% or the aminoacid sequence of above homology, and have α 1, the active protein of 6-fucosyltransferase;
(f) contain with aminoacid sequence shown in the SEQ ID NO:5 and have 80% or the aminoacid sequence of above homology, and have α 1, the active protein of 6-fucosyltransferase.
(7) according to (1) to (6) cell in each, this cell has resistance to the lectin of identification sugar chain, in the said sugar chain 1 of Fucose with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end pass through α-key connection.
(8) according to the cell of (7), at least one lectin of this cell following to being selected from (a) to (d) has resistance:
(a) LcA;
(b) pisum sativum agglutinin;
(c) broad bean lectin;
(d) dried orange peel auricularia auriculajudae lectin.
(9) according to (1) to (8) cell in each, this cell is selected from following (a) to (j):
(a) derive from the Chinese hamster ovary celI of Chinese hamster ovary tissue;
(b) rat myeloma cell is the YB2/3HL.P2.G11.16Ag.20 cell;
(c) mouse myeloma cell line NS0 cell;
(d) mouse myeloma cell line SP2/0-Ag14 cell;
(e) derive from the bhk cell of Syria hamster nephridial tissue;
(f) hybridoma of generation antibody;
(g) human leukemia cell line Namalwa cell;
(h) embryonic stem cell;
(i) fertilized egg cell;
(j) vegetable cell.
(10) according to (1) to (9) cell in each, this cell contains the gene of encoding antibody molecule.
(11) according to the cell of (10), this antibody molecule is selected from following (a) to (d):
(a) people's antibody;
(b) humanized antibody;
(c) contain (a) or (b) antibody fragment in Fc district;
(d) contain (a) or (b) fused protein in Fc district.
(12) according to the cell of (10) or (11), wherein antibody molecule belongs to the IgG type.
(13) according to (1) to (12) cell in each, the antibody compositions that this cell produces has higher antibody dependent cellular mediated cell cytotoxic activity than the antibody compositions of its parental cell generation.
(14) cell of basis (13); The antibody compositions that wherein has a higher antibody dependent cellular mediated cell cytotoxic activity has higher sugar chain ratio than the antibody compositions that its parental cell produces, in the said sugar chain Fucose not with the total complex body of sugar chain that Fc district bonded N-glucosides is connected in the N-acetylglucosamine connection of reducing end.
(15) according to the cell of (14), wherein do not connect in the sugar chain of Fucose, 1 of Fucose not with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end through α-key connection.
(16) according to (13) to (15) cell in each, wherein Fucose not with sugar chain ratio that the N-acetylglucosamine of reducing end is connected through α-key be connected with Fc district bonded N-glucosides in the antibody compositions the total complex body of sugar chain 20% or more than.
(17) according to (13) to (16) cell in each, the antibody compositions that wherein has higher antibody dependent cellular mediated cell cytotoxic activity does not have the N-acetylglucosamine bonded sugar chain of Fucose and sugar chain reducing end.
(18) a kind of method that produces antibody compositions, wherein said method comprise to be used according to asking 10 to 17 cells described in each.
(19) a kind of method that produces antibody compositions, said method are included in the substratum cultivates according to (10) to (18) cell described in each, in substratum, forms and the accumulation antibody compositions; And from substratum, reclaim antibody compositions.
(20) according to (18) or (19) described method, said antibody compositions is the antibody compositions that has higher antibody dependent cellular mediated cell cytotoxic activity than the antibody compositions that its parental cell produces.
(21) step of basis (20); Described antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity has higher sugar chain ratio than the antibody compositions that its parental cell produces, in the said sugar chain Fucose not with the total complex body of sugar chain that Fc district bonded N-glucosides is connected in the N-acetylglucosamine connection of reducing end.Sugar does not combine at reducing end with the N-acetylglucosamine.
(22) according to the step of (21), in the described sugar chain that does not connect Fucose, in 1 total complex body of sugar chain that is not connected of Fucose with the N-glucosides 6 of the N-acetylglucosamine of reducing end through α-key connection.
(23) according to (20) to (22) cell in each; In the total complex body of sugar chain that described antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity is with bonded N-glucosides in antibody compositions Fc district is connected, Fucose not with sugar chain ratio that the N-acetylglucosamine of reducing end is connected through α-key be connected with Fc district bonded N-glucosides in the antibody compositions the total complex body of sugar chain 20% or more than.
(24) according to (20) to (23) cell in each, described antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity is not have the antibody compositions that reducing end N-acetylglucosamine is connected in Fucose and the sugar chain.
(25) use according to (1) to (9) transgenic nonhuman animal that cell produces in each or plant or generation thereafter.
(26) transgenic nonhuman animal of basis (24) or plant or generation thereafter, described transgenic nonhuman animal is selected from ox, sheep, goat, pig, horse, mouse, rat, poultry, monkey and rabbit.
(27) basis (25) or (26) described transgenic nonhuman animal or plant or generation thereafter, it has been imported into the gene of encoding antibody molecule.
(28) transgenic nonhuman animal of basis (27) or plant or generation thereafter, described antibody molecule is selected from following (a) to (d):
(a) people's antibody;
(b) humanized antibody;
(c) contain (a) or (b) antibody fragment in Fc district;
(d) contain (a) or (b) fused protein in Fc district.
(29) transgenic nonhuman animal of basis (27) or (28) or plant or generation thereafter, described antibody molecule belongs to IgG family.
(30) a kind of step that produces antibody compositions is characterized in that comprising and cultivates according to (27) to (29) transgenic nonhuman animal or the plant in each; Separation inductive from cultured animals or plant contains the tissue or the body fluid of antibody molecule; From isolated tissue or body fluid, reclaim the antibody compositions that contains required antibody molecule.
(31) a kind of step that produces antibody compositions, it is characterized in that comprising from according to (26) to (29) each transgenic nonhuman animal or plant or separate the cell that produces antibody in generation thereafter; The cell of the generation antibody of culture of isolated in substratum forms in substratum and the accumulation antibody compositions; From substratum, reclaim antibody compositions.
(32) according to the step of (30) or (31), described antibody compositions is the antibody compositions that has higher antibody dependent cellular mediated cell cytotoxic activity than the antibody compositions of the not adorned transgenic nonhuman animal of genome or plant or generation generation thereafter.
(33) step of basis (32); Described antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity than the not adorned transgenic nonhuman animal of genome or plant or thereafter generation the antibody compositions that produces sugar chain with higher proportion, in the said sugar chain Fucose not with the N-acetylglucosamine connection of the reducing end of the total complex body of sugar chain that is connected with antibody compositions Fc district bonded N-glucosides.
(34) according to the step of (33), 6 of the N-acetylglucosamine of the reducing end position of 1 of Fucose total complex body of sugar chain that is not connected with the N-glucosides are passed through α-key connection in the sugar chain.
(35) according to (32) to (34) step in each, have Fucose in the antibody compositions of higher antibody dependent cellular mediated cell cytotoxic activity not with sugar chain ratio that the N-acetylglucosamine of reducing end is connected through α-key be connected with Fc district bonded N-glucosides in the antibody compositions the total complex body of sugar chain 20% or more than.
(36) according to (32) to (35) cell in each, the antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity is the antibody compositions that does not have Fucose to be connected with the N-acetylglucosamine of reducing end in the sugar chain.
(37) a kind of Fc of containing district has the antibody compositions that the N-glucosides connects the antibody molecule of sugar chain, have Fucose not with the sugar chain of the N-acetylglucosamine connection of the reducing end of the total complex body of sugar chain that is connected with antibody compositions Fc district bonded N-glucosides.
(38) according to 37 described antibody compositions, the sugar chain that does not connect Fucose is 6 of the N-acetylglucosamine sugar chains that connect through α-key of reducing end of 1 sugar chain complex body that is not connected with the N-glucosides of Fucose.
(39) a kind of antibody compositions through (18) to (24) generating step in each.
(40) a kind of antibody compositions through (27) to (36) generating step in each.
(41) contain the medicine of the antibody compositions of with good grounds (37) to (40) in each as activeconstituents.
(42) according to the medicine of (41), it is diagnostic reagent, preventive or the therapeutical agent of following disease: tumour relative disease, transformation reactions relative disease, inflammation related disease, autoimmune disorder, cardiovascular disorder, virus infection relative disease or infectation of bacteria relative disease.
(43) produce according in the medicine of (41) or (42) to according to (37) to (40) each the application of antibody compositions.
In the modifying factor group so that the method that has a modifying factor group in the active cell lower or that removed relevant sugar chain modified enzyme (after this being called " cell of the present invention ") than its parental cell is not specially limited; As long as the modification of cellular genome is (to be called after this that " α 1; the activity of 6-Fucose modifying enzyme "), and the 1-position that it is characterized in that Fucose in the said sugar chain is connected the 6-position of the N-acetylglucosamine of sugar chain complex body reduction end and passes through α-key combination with the N-glucosides in order to have lower than its parental cell or to have removed relevant sugar chain modified enzyme.
Parental cell is to reduce or removal α 1, and the method for 6-Fucose modification enzyme activity is applied to the cell before the genome.Parental cell is restriction especially not, comprises following cell.
The NS0 parental cell comprises NS0 cell such as the BIO/TECHNOLOGY described in the following document, 10, 169 (1992) and Biotechnol.Bioeng., 73, 261 (2001), in RIKEN cell bank, the NSO clone of registering in the physio-chemical study association (RCB 0213) is through migrating to these clones in ubcellular system and the similar cell that obtains in the substratum that they can grow.
The SP2/0-Ag14 parental cell is included in the SP2/0-Ag14 cell described in the following document, like J.Immunol. (Journal of Immunology), 126,317 (1981), Nature (nature), 276.269 (1978) and Human Antibodies and Hybridomas, 3, 129 (1992), the SP2/0-Ag14 cell of in ATCC, registering (ATCC CRL-1581) is through ubcellular system (ATCC CRL-1581.1) and the similar cell that obtains in the substratum that these clones is migrated to their growths.
Be included in the Chinese hamster ovary celI described in the following document from the CHO parental cell of Chinese hamster ovary tissue, like Journal of Experimental Medicine (The Journal of Experimental Medicine) (Jikken Igaku), 108, 945 (1958), Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 60,1275 (1968), Genetics (genetics), 55, 513 (1968), Chromosoma (karyomit(e)), 41., 129 (1973), Methods in Cell Science (cytology method), 18, 115 (1996), Radiation Research (radiological study), 148,260 (1997), Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 77, 4216 (1980), Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 60, 1275 (1968), Cell (cell), 6121 (1975) and Molecular Cell Genetics (genetics), Appendix I, H (883-900 page or leaf); Clone CHO-K1 (ATCC CCL-61), clone DUXB11 (ATCCCRL-9096) and clone Pro-5 (ATCC CRL-1781) in the ATCC registration; Commercially available clone CHO-S (Cat#11619 of Life Technologies) is through ubcellular system and the similar cell that obtains in the substratum that these clones is migrated to their growths.
Rat myeloma cell is that the YB2/3HL.P2.G11.16Ag.20 parental cell comprises the clone of setting up from Y3/Agl.2.3 cell (ATCC CRL-1631), the YB2/3HL.P2.G11.16Ag.20 cell such as the J.Cell.Biol. that describe in the document below, 93, 576 (1982) and Methods Enzymol. (Enzymology method), 73B, 1 (1981), at the YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL-1662) of ATCC registration, through ubcellular system and the similar cell that obtains in the substratum that these clones is migrated to their growths.
16 bonded reaction that are connected the reduction end N-acetylglucosamine of sugar chain through α-key with complex body N-glucosides of Fucose.Relating to the reduction end that connects sugar chain at complex body N-glucosides 1 enzyme with 6 bonded reactions of N-acetylglucosamine through α-key Fucose comprises the reduction end that is connected sugar chain at complex body N-glucosides is reacted influential enzyme through 1 of α-key Fucose and 6 bonded of N-acetylglucosamine.
1,6-Fucose modifying enzyme comprises α 1,6-fucosyltransferase, alpha-L-fucosidase and similar enzyme.
The reduction end that connects sugar chain at complex body N-glucosides through α-key Fucose 1 and 6 bonded of N-acetylglucosamine are reacted influential enzyme also comprise and pass through the influential enzyme of enzymic activity of 6 association reactions of 1 of α-key Fucose and N-acetylglucosamine, with structure influential enzyme as the substrate of enzyme material to relating to the reduction end that is connected sugar chain at complex body N-glucosides.
In the present invention, α 1, and 6-Fucose modifying enzyme comprises following (a), (b), and (c) or the albumen of dna encoding (d) and following (e), (f), (g), (h), (i) or protein (j):
(a) contain the DNA of the nucleotide sequence of SEQ ID NO:1 representative;
(b) contain the DNA of the nucleotide sequence of SEQ ID NO:2 representative;
(c) DNA hybridize under stringent condition and the coding with the nucleotide sequence that contains SEQ ID NO:1 representative has α 1, the active protein DNA of 6-fucosyl transferase;
(d) DNA hybridize under stringent condition and the coding with the nucleotide sequence that contains SEQ ID NO:2 representative has α 1, the active protein DNA of 6-fucosyl transferase;
(e) contain the protein of the aminoacid sequence of SEQ ID NO:4 representative;
(f) contain the protein of the aminoacid sequence of SEQ ID NO:5 representative;
(g) contain the protein of following aminoacid sequence, wherein by the aminoacid sequence of SEQ ID NO:4 representative wherein at least one amino acid deleted, replaced, inserted and/or added, and have α 1, the 6-fucosyl transferase is active;
(h) contain the protein of following aminoacid sequence, wherein at the aminoacid sequence by SEQ ID NO:5 representative, wherein at least one amino acid is deleted, is replaced, is inserted and/or added, and has α 1, and the 6-fucosyl transferase is active;
(i) contain aminoacid sequence with SEQ ID NO:4 representative and have 80% or the aminoacid sequence of more homologys, and have α 1, the active protein of 6-fucosyl transferase;
(j) contain aminoacid sequence with SEQ ID NO:5 representative and have 80% or the aminoacid sequence of more homologys, and have α 1, the active protein of 6-fucosyl transferase, and analogue.
In the present invention; The DNA of hybridize under stringent condition for example passes through like clone hybridization (colony hybridization); The method of plaque hybridization or Southern blot hybridization; Use as have DNA or the DNA that its part fragment obtains as probe of the nucleotide sequence of SEQ ID NO:1 or 2 representatives, and particularly including can be in the presence of 0.7 to 1.0M sodium-chlor, the employing filter membrane be hybridized the DNA that is identified at 65 ℃; Wherein the dna fragmentation in colony or plaque source is fixed on the filter membrane, uses 0.1 to 2 * SSC solution (compsn of 1 * SSC solution of being made up of the Trisodium Citrate of the sodium-chlor of 150mM and 15mM) to wash filter membrane at 65 ℃ then.Can hybridize according to the method for having described, as at Molecular Cloning, A Labooratory Manual (molecular cloning; Laboratory manual), second edition, Cold Spring Harbor Laboratory Press (cold spring harbor laboratory's printing) (1989) (after this being called " molecular cloning; second edition "), modern molecular biology method, John Wiley & Sons; 1987-1997 (after this being called " modern molecular biology method "), dna clone: core technology, hands-on approach; Second edition, Oxford (1995); With similar article.Interfertile DNA comprises with nucleotide sequence shown in SEQIDNO:1 or 2 having at least 60% or more, preferred 70% or more, more preferably 80% or more, further preferred 90% or more, more preferably 95% or more, most preferably 98% or more.
In the present invention, contain the aminoacid sequence of SEQ ID NO:4 or 5 representatives, wherein at least one amino acid is deleted; Replace, insert and/or interpolation, and have α 1; The active protein of 6-fucosyl transferase can have respectively in the proteinic coding DNA of aminoacid sequence shown in SEQ ID NO:4 or 5 through for example a site directed mutation being imported, and uses the site directed mutation that is described below; As at molecular cloning, second edition; The modern molecular biology method, Nucleic Acids Research (nucleic acids research), 10, 6487 (1982); Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 79, 6409 (1982); Gene (gene), 34, 315 (1985); Nucleic Acids Research (nucleic acids research), 13, 4431 (1985); Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 82, 488 (1985); With similar article.Deleted, replace, insertion and/or the amino acid quantity of adding are one or more; This quantity is restriction especially not, but this quantity is to delete like site directed mutation through a kind of known technology; Replace or add, for example 1 to tens, preferred 1 to 20; More preferably 1 to 10, most preferably 1 to 5.
In the present invention, contain with aminoacid sequence shown in SEQ ID NO:4 or 5 and have 80% or the aminoacid sequence of more homologys, and have α 1, the active protein of 6-fucosyltransferase, when adopt analysis software such as BLAST [J.Mol.Biol., 215, 403 (1990)], FAST A [Enzymology method, 183, 63 (1990)] or similar approach when calculating, have at least 80% or more with aminoacid sequence shown in SEQ ID NO:4 or 5; Preferred 85% or more, more preferably 90% or more, still more preferably 95% or more; More preferred 97% or more, most preferably 99% or more homology.
In the present invention; The modifying factor group is so that the reduction of relevant sugar chain modified enzymic activity or eliminate and surpass its parental cell; 1 of Fucose 6 connection that are connected the reduction end N-acetylglucosamine of sugar chain through α-key with complex body N-glucosides in the described sugar chain; Its implication is that sudden change is imported into the expression control region of enzyme, so that reduce the expression of this kind of enzyme, or sudden change is imported in the aminoacid sequence of gene so that reduce the function of this kind of enzyme.The implication of importing sudden change is in genomic nucleotide sequence, to modify, and like deletion, replaces, and inserts and/or interpolation.
The implication of the cell that genomic gene is knocked out is that the expression or the function of genomic gene in cell is suppressed fully.The cell that genomic gene is knocked out comprises the cell of from genome, deleting goal gene wholly or in part.As the method that obtains this cell, can use any technology, as long as the goal gene group can be modified.But preferred gene engineering.Example comprises:
(a) a kind of gene disruption technology comprises target coding 1, the gene of 6-Fucose modifying enzyme,
(b) import coding 1, the technology of the negative mutator gene of 6-Fucose modifying enzyme dominance,
(c) will suddenly change and import coding 1, the technology in the gene of 6-Fucose modifying enzyme,
(d) suppress coding for alpha 1, the genetic transcription of 6-Fucose modifying enzyme and/or the technology of translation.
And; Cell of the present invention can obtain through the method below using; Promptly through use selecting phytohemagglutinin is had the clone of resistance, said lectin can be discerned 1 of Fucose wherein is connected the reduction end N-acetylglucosamine of sugar chain with complex body N-glucosides through α-key the sugar chain of 6 connections.
The growth of phytohemagglutinin resistant cell does not receive the inhibition of phytohemagglutinin effective concentration in the cell cultivation process.Effective concentration is the concentration that parental cell can not normal growth; Or be higher than the concentration that this concentration parental cell can not be grown; This concentration preferred class is similar to the concentration that parental cell can not normal growth; Being more preferably 2 to 5 times of the concentration that parental cell can not normal growth, still more preferably is 10 times of the concentration that parental cell can not normal growth at least, most preferably is higher than at least 20 times of the concentration that parental cell can not normal growth.
In the present invention, the effective concentration that can not suppress the growing plants lectin can decide according to clone, is generally 10 μ g/ml to 10.0mg/ml, and preferred 0.5 to 2.0mg/ml.
As lectin, can use any lectin, as long as it is to discern 1 of Fucose is incorporated into the compound sugar chain reducing end under neutral of N-glucosidic linkage through the α key the lectin of sugar chain of 6 of N-acetyl-glucosamines.Example comprises LcA LCA (deriving from the LCA of Lens culinaris); Pisum sativum agglutinin PSA (deriving from the pisum sativum agglutinin of Pisum sativum); A kind of broad bean lectin VFA (deriving from the lectin of Vicia faba), dried orange peel auricularia auriculajudae lectin AAL (deriving from the lectin of Aleuria aurantia) or the like.
Cell of the present invention can be any cell, as long as it can express a kind of antibody molecule.Example comprises yeast, zooblast, insect cell, vegetable cell and analogue, and concrete example is included in those described in following the 3rd.Zooblast comprises the Chinese hamster ovary celI from the Chinese hamster ovary tissue, and rat myeloma cell is the YB2/3HL.P2Gl1.16Ag.20 cell, mouse myeloma cell line NSO cell; Mouse myeloma SP2/0-Agl4 cell; From the BHK cell of Syria hamster nephridial tissue, produce the hybridoma of antibody, human leukemia cell line Namalwa cell; Embryonic stem cell, fertilized egg cell and similar cell.Preferred example comprises above-mentioned myeloma cell and the hybridoma that is used to produce antibody compositions; Produce the host cell of humanized antibody and people's antibody; Preparation can produce the non-human transgenic animal's of people's antibody embryonic stem cell and fertilized egg cell; Preparation can produce vegetable cell and the similar cell of the transgenic plant of humanized antibody and people's antibody.
The antibody compositions that cell of the present invention produces is higher than the ADCC activity of the antibody compositions that parental cell produced.
And; Cell of the present invention can produce a kind of antibody compositions; Wherein with compsn in Fc district bonded N-glucosides be connected in the total complex body of sugar chain, Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio to produce antibody compositions than parental cell high.
The present invention relates to a kind of process that produces antibody compositions, it is characterized in that using cell of the present invention.
Antibody compositions is that a kind of Fc of containing district has the compsn that the N-glucosides connects the antibody molecule of sugar chain complex body.
Antibody is a kind of tetramer, two polypeptied chains wherein, and heavy chain (after this being called " H chain ") links to each other respectively with two molecules in the light chain (after this being called " L chain ").What about 1/4th and L chain N-of each H chain N-end was terminal about 1/4th (each all surpasses 100 amino acid) is called the V district, and this district is rich in variety, and directly relevant with antigenic combination.The V district is called the C district with the major part of outside part.According to the homology in C district, antibody molecule is divided into IgG, IgM, IgA, several types of IgD and IgE.
Equally, the homology IgG type according to the C district also further is divided into IgG1 to IgG4 hypotype.
The H chain is divided into four immunoglobulin domain VH from its N-end side, CH1, and CH2 and CH3, and between CH1 and CH2, CH1 and CH2 divided the peptide zone of the alterable height of opening, be called hinge area.CH2 and the structure unit of CH3 contained behind the hinge area are called the Fc district, and the N-glucosides connects above sugar chain is combined in, and this district also is the Fc acceptor, complement and analogue bonded regional (the immunology atlas, master, Nankodo, on February 10th, 2000 issued, the 5th edition; Antibody technique handbook (Kotai Kogaku Nyumon), Chijin Shokan, on January 25th, 1994, first version).
According to the combining form of protein portion; Gp; Sugar chain like antibody is divided into two types roughly, promptly with l-asparagine (sugar chain that the N-glucosides is connected) bonded sugar chain and with other amino acid, like Serine, Threonine (O-glycosides connection sugar chain) bonded sugar chain.The N-glucosides connects sugar chain has a basic common core structure, representes [method (Gakujutsu Shuppan center) of Biochemistry Experiment method 23-research glycoprotein candy chain, Reiko Takahashi edits (1989)] by following structural formula (I):
In structural formula (I), be called reduction end with l-asparagine bonded sugar-chain end, an opposite side is called non-reduced end.
It can be that any N-glucosides connects sugar chain that the N-glucosides connects sugar chain, as long as it contains the core texture of structural formula (I).Example comprises high mannose type, and wherein seminose combines separately with the non-reduced end of core texture; The mixture type; Wherein the non-reduced end side of core texture has a semi-lactosi-N-acetylglucosamine (after this being called " Gal-GlcNAc ") at least; The non-reduced end side of Gal-GlcNAc has a kind of sialyl, five equilibrium N-acetylglucosamine or similar structure; The hybridization type, wherein the non-reduced end side of core texture contains two kinds of branches of high mannose type and mixture type; Or the like.
Connect sugar chain independence bonded position because the Fc district in the antibody molecule has the N-glucosides, two sugar chains combine an antibody molecule.Comprise any sugar chain that contains core texture shown in the structural formula (I) because be connected sugar chain, be connected the combination that sugar chain has many sugar chains with two N-glucosides of antibodies with antibody molecule bonded N-glucosides.
Therefore, in the present invention, use the antibody compositions of the present invention of cell preparation of the present invention, contain the antibody of identical sugar chain structure or different sugar chain structure, as long as can obtain effect of the present invention from said composition.A kind of antibody compositions below antibody compositions of the present invention is preferred; Wherein with antibody compositions in be connected in the total complex body of sugar chain with Fc district bonded N-glucosides, Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio high than the genome antibody compositions that adorned parental cell did not produce.
And; In the present invention; Antibody compositions through using non-human animal or plant or generation preparation thereafter contains the antibody with identical sugar chain structure or different sugar chain structure, as long as can obtain effect of the present invention from said composition, it is characterized in that said animal or plant or in generation thereafter; Genome is modified so that its α 1, and the activity of 6-Fucose modifying enzyme reduces more or is eliminated.
A kind of antibody compositions below antibody compositions of the present invention is preferred; Wherein with antibody compositions in Fc district bonded N-glucosides be connected in the total complex body of sugar chain, Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio through use not adorned non-human animal of genome or plant or thereafter generation (after this being called " parental generation is individual ") the antibody compositions height that produced.
Genome is modified so that its α 1, the activity of 6-Fucose modifying enzyme reduce more the transgenic nonhuman animal that is eliminated or plant or thereafter generation can use embryonic stem cell, zygote or vegetable cell prepare.
In the present invention; With antibody compositions in the Fc district bonded N-glucosides that comprises be connected in the total complex body of sugar chain, Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio be wherein Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain quantity with antibody compositions in the Fc district bonded N-glucosides that comprises be connected the ratio of sugar chain complex body total quantity.
Wherein be not connected in the sugar chain complex body reduction end N-acetylglucosamine bonded sugar chain with the N-glucosides be to connect in the sugar chain complex body at the N-glucosides to Fucose, Fucose not with reduction end in the N-acetylglucosamine through α-key bonded sugar chain.In particular, wherein the 1-position of Fucose is not connected the sugar chain complex body with the 6-position of N-acetylglucosamine through α-key bonded N-glucosides.
With antibody compositions of the present invention in the Fc district bonded N-glucosides that comprises be connected in the total complex body of sugar chain; Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio preferred 20% or more; More preferably 30% or more; Still more preferably 40% or more, most preferably 50% or more, even most preferably 100%.
There is the active antibody compositions of higher ADCC to comprise than parental cell or the individual antibody compositions that produces of parental generation; With antibody compositions in Fc district bonded N-glucosides be connected in the total complex body of sugar chain, Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio high than the individual antibody compositions that is produced of parental cell or parental generation.Example comprises a kind of antibody compositions, and its activity is at least 2 times, preferably at least 3 times, and more preferably at least 5 times, further preferred 10 times or higher.Most preferred antibody compositions be all N-glucosides of wherein being connected to Fc district in the antibody compositions connect sugar chain complex bodys be wherein Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain.
In the sugar chain Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio be 100% antibody compositions or all N-glucosides be connected that the sugar chain complex body all is connected to Fc district in the antibody compositions be Fucose the 1-position not with reduction end in the 6-position bonded sugar chain of N-acetylglucosamine, Fucose wherein can not detect through the 5th following said sugar chain analytical procedure.
In the antibody compositions that the present invention obtains; Be connected in the total complex body of sugar chain with Fc district bonded N-glucosides; Wherein Fucose not with the sugar chain reduction end in N-acetylglucosamine bonded sugar chain ratio high than the individual antibody compositions that is produced of parental cell or parental generation, the antibody compositions that the ADCC activity of the antibody compositions that obtains among the present invention contains parental cell or the individual antibody molecule that produces of parental generation is high.
The ADCC activity is a kind of cytotoxic activity; With the cell-surface antigens bonded antibody that exists on the tumour cell in vivo can be through being present in the Fc receptor activation effector cell who exists on antibody Fc district and the effector cell surface; Therefore can disturb [monoclonal antibodies: principle and application such as tumour cell; Wiley-Liss, Inc., 2.1 chapters (1955)].The effector cell comprises killer cell, nk cell, activatory scavenger cell etc.
Wherein Fucose not with compsn in the sugar chain reduction end that comprises the mensuration of N-acetylglucosamine bonded sugar chain ratio can adopt known method such as hydrazinolysis; Enzymic digestion or similar approach discharge sugar chain from antibody molecule; It is characterized in that described compsn contains antibody molecule [Biochemical Experimentation Methods 23-Method for Studying Glyoprotein Sugar Chain (the Biochemistry Experiment method with N-glucosides connection sugar chain complex body in the Fc district; The method 23 of the sugar chain structure of research gp) (Japan Scientific Societies Press (Japanese science association publishes)); Reiko Takahashi writes (1989)] the release sugar chain is measured from antibody molecule; Sugar chain for discharging carries out fluorescent mark or radio-labeling, passes through the sugar chain of chromatography method separation marking then.Selectable, the sugar chain of release also can confirm with the HPAED-PAD methods analyst, [J Liq Chromatogr., 6,1577 (1983)].
Equally; Antibody of the present invention preferably can be discerned the antibody of taa; The virus of being given an example below the antibody of identification transformation reactions or inflammation related antigen, the antibody of identification cardiovascular disorder related antigen or identification or the antibody of infectation of bacteria related antigen preferably belong to the IgG type.
Identification taa antibody comprise anti--GD2 antibody [Anticancer Res. (anticancer research), 13, 331-336 (1993)], anti--GD3 antibody [Cancer Immunol.Immunother., 36, 260-266 (1993)], anti--GM2 antibody [Cancer Res. (cancer research), 54, 1511-1516 (1994)], anti--HER2 antibody [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 89, 4285-4289 (1992)], anti-CD 52 antibody [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 89, 4285-4289 (1992)], anti--MAGE antibody [British J.Cancer (cancer), 83, 493-497 (2000)], anti--HM1.24 antibody [Molecular Immunol. (molecular immunology), 36, 387-395 (1999)], anti--parathyroid hormone-related protein (PTHrP) antibody [Cancer (cancer), 88, 2909-2911 (2000)], alkali-resistivity fibroblast growth factor antibody and anti--FGF8 antibody [Proc.Nail.Acad, Sci.USA, 86, 9911-9915 (1989)], alkali-resistivity fibroblast growth factor receptor antibody and anti--FGF8 receptor antibody [J.Biol.Chem. (journal of biological chemistry), 265,16455-16463 (1990)], synalbumin like growth factor antibody [J.Neurosci.Res., 40, 647-659 (1995)], anti-IGFR body antibody [J.Neurosci.Res., 40, 647-659 (1995)], anti--PMSA antibody [J.Urology (urology research), 160.2396-2401 (1998)], anti-vascular endothelial cell growth factor antibody [Cancer Res. (cancer research), 57,4593-4599 (1997)], anti-vascular endothelial cell growth factor receptor 2 body antibody [OncoGene (gene), 19, 2138-2146 (2000)], anti--CA125 antibody, anti--17-1A antibody; Anti-integrin alpha v beta 3 antibody, anti--CD33 antibody, anti--CD22 antibody; Anti--hla antibody, anti--HLA-DR antibody, anti-CD 20 antibodies; Anti--CD19 antibody, anti--EGF receptor antibody [Immunology Today (immunology today) 21(8), 403-410 (2000)], anti--CD 10 antibody [American Journal of Clinical Pathology (U.S.'s clinical pathology magazine), 113, 374-382 (2000)] etc.
The antibody of identification transformation reactions or inflammation related antigen comprise anti-interleukin 6 antibody [Immunol.Rev. (immunology research), 127, 5-24 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol. (molecular immunology), 31, 371-381 (1994)], anti-interleukin-15 antibody [Immunol.Rev (immunology research)., 127, 5-24 (1992)], anti-interleukin-15 receptor antibody and anti-interleukin 4 antibody [Cytokine (cytokine), 3, 562-567 (1991)], anti-interleukin 4 antibody [J.Immunol.Meth. (immunological method magazine), 217, 41-50 (1991)], D2E7 [Hybridoma, 13, 183-190 (1994)], the anti-tumor necrosis factor receptor antibody [Molecular PharmacoL, 58, 237-245 (2000)], anti--CCR4 antibody [Nature (nature), 400,776-780 (1999)], anti-chemokine antibody [J.Immuno.Meth. (immunological method magazine), 174,249-257 (1994)], anti-chemokine receptor antibody [J.Exp.Med. (experimental technique magazine), 186,1373-1381 (1997)], anti--IgE antibody, anti--CD23 antibody, anti--CD 11a antibody [Immunology Today (immunology today), 21(8), 403-410 (2000)], anti--CRTH2 antibody [J.Immuno. (Journal of Immunology), 162,1278-1286 (1999)], anti--CCR8 antibody (WO99/25734), anti--CCR3 antibody (US6207155) or the like.
The antibody of identification cardiovascular disorder related antigen comprise anti--GpIIb/IIIa antibody [J.Immunol. (Journal of Immunology), 152,2968-2976 (1994)], antiplatelet derivative growth factor antibody [Science (science), 253,1129-1132 (1991)], antiplatelet derived growth factor receptor antibody [J.Biol.Chem. (journal of biological chemistry), 272,17400-17404 (1997)], anticoagulin antibody [Circulation (circulation), 101,1158-1164 (2000)] or the like.
The antibody of identification autoimmune disease related antigen comprise anti-from the body dna antibody [Immunol.Letters, 72, 61-68 (2000)] or the like.
The antibody of identification virus or infectation of bacteria related antigen comprise anti-gp120 antibody [Structure (structure), 8, 385-395 (2000)], anti-CD 4 antibodies [J.Rheumatology (rheumatology magazine), 25, 2065-2076 (1998)], anti--CCR4 antibody, anti--Vero toxin antibody [J.Clin.Microbiol, 37396-399 (1999)]; The antibody of autoimmune disorder (psoriatic, rheumatic arthritis, clone disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis; Deng), anti--CD11a antibody, anti--ICAM3 antibody, anti--CD80 antibody, anti--CD2 antibody, anti-CD 3 antibodies, anti-CD 4 antibodies, anti--integrin alpha 4 β 7 antibody, anti--GD40L antibody, anti--IL-2 antibody [Immunology Today (immunology today) 21(8), 403-410 (2000)], or the like.
Antibody molecule can any antibody molecule, as long as it contains the Fc district of antibody.Example comprises antibody, and antibody fragment contains the fusion rotein in Fc district, or the like.
Antibody is a kind of protein that stimulates the immunity system generation that in live body, causes as exogenous antigen, has and antigen-specific bonded activity.Example comprises by the hybridoma excretory antibody with the splenocyte preparation of the animal of antigen immune; The antibody of genetic recombination techniques preparation just imports the antibody that obtains in the host cell through the antibody expression vector that will insert antibody gene; Or the like.Concrete example comprises the antibody that is produced by hybridoma, humanized antibody, people's antibody or the like.
Hybridoma is to use from the B cell of the non-human mammal of antigen immune and a kind of cell that obtains from cytogamy between the myeloma cell in mouse or similar source, can produce the monoclonal antibody with required antigen-specific.
Humanized antibody comprises people's chimeric antibody, people's CDR grafted antibody etc.
People's chimeric antibody be the variable region of heavy chain that comprises the non-human antibody (below be called " HV " perhaps " VH "; Variable chains and heavy chain are respectively " V zone " and " H chain ") and the variable region of non-human antibody's light chain (below be called " LV " perhaps " VL "), the antibody of human antibody heavy chain's constant region (be also referred to as be " CH " after this) and human antibody light chain constant region (be also referred to as be " CL " after this).For the non-human animal, any animal is mouse for example, rat, and hamster, rabbits etc. can be used, as long as can therefrom prepare hybridoma.
CDNA through preparation coding VH and VL from the hybridoma that produces monoclonal antibody; Insert the expression vector that is used for host cell to them; Host cell has the gene of coding human antibody CH and people's antibody CL; The carrier of construction expression people chimeric antibody imports the host cell that is used for expression vector to carrier then, produces people's chimeric antibody.
For the CH of people's chimeric antibody, can use any CH, as long as it belongs to Tegeline (below be called " hIg ") of people.Those classifications that belong to hIgG are preferred, also can use the subclass of any hIgG of belonging to class, like hIgG1, and hIgG2, hIgG3 and hIgG4.Likewise,, can use any CL,, and also can use those to belong to the CL of κ or λ class as long as it belongs to the hIg class for the CL of people's chimeric antibody.
People CDR-grafted antibody is the antibody of the correct position of the aminoacid sequence of CDR of VH and the VL of the wherein nonhuman animal antibody VH and the VL that are transplanted to people's antibody.
People CDR-grafted antibody can be through making up the cDNA preparation in coding V zone; The CDR that wherein derives from non-human animal's antibody VH and VL is transplanted to the CDR zone of the VH and the VL of people's antibody; They are inserted into the expression vector that is used for host cell; This host cell contains the gene of coding human antibody CH and people's antibody CL, has made up the carrier of the expression that is used for people-CDR grafted antibody thus, then expression vector is imported to host cell and comes expressing human CDR-grafted antibody.
For the CH of people CDR-grafted antibody, can use any CH, as long as it belongs to the hIg class, but hIgG is preferred, any subclass that belongs to the hIgG class, like hIgG1, hIgG2, hIgG3 and hIgG4 also can use.Likewise,, can use any CL,, and also can use those to belong to the CL of κ or λ class as long as it belongs to the hIg class for the CL of people CDR-grafted antibody.
Originally people's antibody be the antibody that exists naturally in the human body, but also comprise from people's antibody phage storehouse, produces the transgenic animal of people's antibody and produce the antibody that obtains in the transgenic plant of people's antibody.Producing the transgenic animal of people's antibody and the transgenic plant of generation people antibody is with genetically engineered, and cell processes is that the basis prepares with the latest developments of growing engineering.
Through the separation of human peripheral blood lymphocyte, make its immortalization with infection such as Epstein-Barr virus, obtain to produce the lymphocyte of antibody to its clone, cultivate this lymphocyte, separation and antibody purification from substratum and the antibody that obtains to exist in the human body.
People's antibody phage storehouse is a kind of library, wherein inserts phage gene through a gene of the encoding antibody that from human B cell, prepares, antibody fragment such as Fab (Fab), and single-chain antibodies etc. are expressed in the surface of phage.Expression has the phage of the antibody fragment of needed antigen-binding activity, can use it to be incorporated into the activity of the substrate of immobilized antigen, serves as a mark and from the library, reclaims.Antibody fragment can further be converted into the human antibody molecules that comprises two total length H chains and total length L chain through recombinant DNA technology.
The transgenic nonhuman animal that produces people's antibody is that people's antibody is imported into the non-human animal in the cell.The transgenic animal that particularly produce people's antibody can advance embryo stem cell transplantation in the body early embryo of other mouse through human immunoglobulin gene being imported in the embryonic stem cell of mouse, it is grown prepare.Through people's chimeric antibody gene is imported in the zygote, and grow, also can prepare transgenic nonhuman animal.With regard to regard to preparation people antibody the transgenic nonhuman animal that produces people's antibody, obtain to produce the hybridoma of people's antibody through using the hybridoma preparation method who carries out at non-human mammal usually, cultivate then, can produce people's antibody and in substratum, accumulate.
Genetically modified non-human animal comprises ox, sheep, goat, pig, horse, mouse, rat, poultry, monkey, rabbit etc.
Also preferred antibody is the antibody that can discern taa in the present invention; The antibody of identification transformation reactions or inflammation related antigen; The antibody of identification cardiovascular disorder related antigen; Discern the antibody of autoimmune disorders related antigen or the antibody of identification virus or infectation of bacteria related antigen, preferably belong to people's antibody of IgG type.
Antibody fragment is the fragment that contains partial antibody Fc district.The Fc district is the zone that is positioned at the heavy chain of antibody C-terminal, comprises natural type and sudden change class.The part in IgG type Fc district is according to people EU exponential such as Kabat numberings, be Cys from C-terminal 226 sites to C-terminal, or from the Pro in C-terminal 230 sites to C-terminal [Sequences of Proteins of Immunological Interest, 5 ThEd., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)].Example comprises antibody, and antibody fragment contains the fusion rotein in Fc district and analogue.Antibody fragment comprises H chain monomer, H chain homodimer and analogue.
The fusion rotein that contains Fc district part is a kind of protein, wherein by the antibody and the protein in the Fc district of antibody or antibody fragment, like a kind of protein (after this being called " Fc fusion rotein ") of enzyme or cytokine fusion.
The present invention is illustrated in detail below.
1. prepare cell of the present invention
Cell of the present invention is through following technology preparation.
(1) contains the gene disruption technology of target codase genomic constitution
Cell of the present invention is through using target coding 1, and the gene disruption technology of the gene of 6-Fucose modifying enzyme prepares, through.The enzyme of relevant sugar chain modified effect comprises α 1, the 6-fucosyltransferase, and alpha-L-fucosidases etc. wherein connect in the sugar chain complex body at the N-glucosides, and the 6-position of N-acetylglucosamine combines through α-key in the 1-position of Fucose and the reduction end.
The gene disruption method can be any method, destroys target enzyme gene as long as it comprises.Example comprises homologous recombination method, and RNA-DNA oligonucleotide (RDO) method adopts retroviral method, uses the method for transposon etc.These methods specifically describe as follows.
(a) prepare antibody produced cell of the present invention through homologous recombination
Cell of the present invention can pass through target coding for alpha 1, and the homologous recombination technique of 6-Fucose modification gene is in the goal gene preparation of modifying on the karyomit(e).
Goal gene on the karyomit(e) can be employed in the operation of mouse embryo; Laboratory manual; Second edition, the method described in the Cold Spring Harbor Laboratory Press (cold spring harbor laboratory's printing) (1994) (after this being called " operation of mouse embryo, laboratory manual ") is modified; Gene target, hands-on approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, gene target adopts ES cell preparation sudden change mouse, Yodo-sha (1995) (after this being called " adopting ES cell preparation sudden change mouse "); Or similar approach, as follows.
Preparation coding for alpha 1, the cDNA of 6-Fucose modifying enzyme.
According to the cDNA that obtains, preparation coding for alpha 1, the genomic dna of 6-Fucose modifying enzyme.
According to the nucleotide sequence of genomic dna, for the homologous recombination system of wanting adorned goal gene is equipped with purpose carrier (for example, α 1, the structure gene of 6-Fucose modifying enzyme, or promoter gene).
The generation of host cell of the present invention can import in the host cell through the purpose carrier with preparation, and selects a kind of cell, wherein between goal gene and purpose carrier, homologous recombination has taken place.
As host cell, can use any cell such as yeast, zooblast, insect cell or vegetable cell are as long as it has coding for alpha 1, the goal gene of 6-Fucose modifying enzyme.Example is included in the cell described in following the 3rd.
Obtain coding for alpha 1, the cDNA of 6-fucosyltransferase or the method for genomic dna comprise the method that is described below.
The preparation method of cDNA:
Total RNA of preparation or mRNA from various host cells.
Prepare the cDNA library from the total RNA or the mRNA of preparation.
With α 1,6-Fucose modifying enzyme is that the basis produces degenerated primer, and the cDNA library of for example adopting preparation obtains coding for alpha 1, the human amino acid sequence and the gene fragment of 6-Fucose modifying enzyme as template through PCR.
Can be through using acquired gene fragment as probe, screening cDNA library obtains coding for alpha 1, the cDNA of 6-Fucose modifying enzyme.
As the mRNA of various cells, can use the commercially available prod (as, make by Clontech) or can from multiple host cell, prepare as follows.From various host cells the preparation total RNA method comprise guanidine thiocyanate-trifluoracetic acid caesium method [Enzymology method, 154,3 (1987)], acid guanidine thiocyanate phenol chloroform (AGPC) method [Analytical Biochemistry (biological chemistry), 162,156 (1987); Experimental Medicine (Jikken Igaku), 9, 1937 (1991)] and similar approach.
And preparation mRNA is poly (A) from total RNA +The method of RNA comprises plain post method (molecular cloning, second edition) of widow (dT)-cured fiber and similar approach.
In addition, use like Fast Track mRNA separating kit (Invitrogen manufacturing), QuickPrep mRNA purification kit (Pharmacia manufacturing) or similar agents box prepare mRNA.
Prepare the cDNA library from people or the non-human animal tissue or the cell mRNA of preparation.The method for preparing the cDNA library is included in molecular cloning, second edition; The modern molecular biology method, laboratory manual, second edition (1989); With the method described in the similar article; Or use the commercial reagent box as carrying out the synthetic Superscript pUC pUC (Life Technologies manufacturing) with plasmid clone of cDNA, the method for ZAP-cDNA synthetic agent box (STRATAGENE (gene) manufacturing) and similar reagents box.
Cloning vector as for preparation cDNA library can use any carrier such as phage vector, plasmid vector or analogue, as long as it can be in e. coli k12 self-replicating.Example comprise ZAP Express [STRATAGENE (gene) makes, Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research (nucleic acids research), 17, 9494 (1989)], Lambda ZAP II (STRATAGENE (gene) manufacturing), λ gt10 and λ gt11 [dna clone; Hands-on approach, 1,49 (1985)], λ TriplEx (Clontech manufacturing); λ ExCell (Pharmacia manufacturing), pcD2 [Mol.Cell.Biol 3, 280 (1983)], PUC18 [Gene (gene), 33, 103 (1985)] and analogue.
Any mikrobe can be used as the host microorganism in preparation cDNA library, preferred intestinal bacteria.Example comprise intestinal bacteria XL1-Blue MRJF ' [STRATAGENE (gene) makes, Strategies, 5, 81 (1992)], intestinal bacteria C600 [Genetics (genetics), 39, 440 (954)], intestinal bacteria Y1088 [Science (science), 222.778 (1983)], intestinal bacteria Y1090 [Science (science), 222.778 (1983)], intestinal bacteria NM522 [J.Mol.Biol, 166,1 (1983)], intestinal bacteria K802 [J.Mol Biol., 16, 118 (1966)], intestinal bacteria JM105 [Gem, 38,275 (1985)] and analogue.
The CDNA library can be used in the analysis subsequently, and is effective as far as possible through the ratio that reduces the long cDNA of infull so that obtain full-length cDNA, the cDNA library that the few cap method of developing through people such as use Sugano prepares [Gene (gene), 138,171 (1994); Gene (gene), 200,149 (1997); Albumen, nucleic acid, albumen, 41, 603 (1996); Experiment medicine (Jikken Igaku), 11, 2491 (1993); CDNA clones (Yodo-sha) (1996); The method (Yodo-sha) (1994) for preparing gene library] can be used in the following analysis.
With α 1; The aminoacid sequence of 6-Fucose modifying enzyme is the basis, and 5 ' the terminal and sequence-specific degenerated primer of 3 ' terminal nucleotide of the nucleotide sequence of encoding amino acid sequence is inferred in preparation, and DNA uses the cDNA library of preparation as template; Through pcr amplification [PCR method; Academic Press (1990)], obtain coding for alpha 1, the gene fragment of 6-Fucose modifying enzyme.
Usually through being used for the method for analysis of nucleotide, as people's such as Sanger dideoxy method [Proc.Nail.Acad.Sci.USA, 74, 5463 (1977)], nucleotide sequence analysis appearance such as ABEPRISM377DNA sequenator (PE Biosystems manufacturing) or similar approach, acquired gene fragment can be proved to be coding for alpha 1, the DNA of 6-Fucose modifying enzyme.
Coding for alpha 1; The DNA of 6-Fucose modifying enzyme can obtain (molecular cloning through carrying out colony hybridization or plaque hybridization; Second edition), use gene fragment as dna probe, the mRNA in being included in people or non-human animal tissue or cell obtains synthetic cDNA or cDNA library.
Coding for alpha 1; The DNA of 6-Fucose modifying enzyme also can obtain through carrying out the PCR screening; MRNA synthetic cDNA or the cDNA library of use from be included in people or non-human animal tissue or cell is as template, and the primer of use can be used to obtain coding for alpha 1, the gene fragment of 6-Fucose modifying enzyme.
Acquired coding for alpha 1, the nucleotide sequence of the DNA of 6-Fucose modifying enzyme is analyzed from its end, measure through the method that is generally used for analysis of nucleotide, as people's such as Sanger dideoxy method [Proc.Nail.Acad, Sci.USA, 74, 5463 (1977)], nucleotide sequence analysis appearance such as ABIPRISM 377DNA sequenator (PE Biosystems manufacturing) or similar approach.
Coding for alpha 1, the gene of 6-Fucose modifying enzyme also can be confirmed by the gene from DB, classify the basis as with the nucleotides sequence of definite cDNA, use homology search program such as BLAST through search nucleotide sequence database such as GenBank, EMBL, DDBJ etc.
Coding for alpha 1, the gene nucleotide series of 6-Fucose modifying enzyme contains the nucleotide sequence shown in SEQ ID NO:1 or 2.
Coding for alpha 1; The cDNA of 6-Fucose modifying enzyme also can obtain through chemosynthesis, classifies the basis as with the nucleotides sequence of definite DNA, uses a kind of dna synthesizer; Like 392 type dna synthesizers of Perkin Elmer manufacturing, or use the chemosynthesis of phosphoramidite method to obtain.
As preparation coding for alpha 1, the method example of the genomic dna of 6-Fucose modifying enzyme, method described below illustrates.
The preparation method of genomic dna:
The method for preparing genomic dna comprises known method, and molecular cloning is seen in its description, second edition; The modern molecular biology method; With similar document.In addition, coding for alpha 1, the genomic dna of 6-Fucose modifying enzyme also can adopt test kit, and Tathagata is carried out genome dna library screening system (Genome Systems manufacturing), general GenomeWalker TMTest kit (CLONTECH manufacturing) or analogue.
Coding for alpha 1, the nucleotide sequence of the genomic dna of 6-Fucose modifying enzyme contains the nucleotide sequence shown in the SEQ ID NO:3.
The purpose carrier that in the homologous recombination of goal gene, uses can prepare according to method described below, like gene target, and hands-on approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, gene target adopts ES cell preparation mutant mice, Yodo-sha (1995); Or similar document.The purpose carrier can use replacement type and insert type.
For the purpose carrier is imported various host cells, can use the method for the importing recombinant vectors of the suitable multiple host cell described in the 3rd below.
Effectively select the method for homologous recombination body to comprise that as just selecting, promotor is selected, negative select or polyA selects, gene target is seen in its description, hands-on approach, ERL Press at Oxford University Press (1993); Biomanual Series 8, gene target adopts ES cell preparation mutant mice, Yodo-sha (1995); Or similar document.The method of selection purpose homologous recombination body comprises the Southern hybrid method (molecular cloning, second edition) of genomic dna, PCR [PCR method, Academic Press (1990)], and similar approach from the clone who selects.
The acquisition of homologous recombination body can be the basis with the activity change of relevant sugar chain modified enzyme, and wherein the 1-position of Fucose combines through α-key with the 6-position of N-acetylglucosamine is connected sugar chain at the N-glucosides reduction end.Following method is described below as the example of the method for selecting transformant.
Select the method for transformant:
Select wherein α 1; The method of the activity reduction of 6-Fucose modifying enzyme or the cell that is eliminated comprises biochemical method or gene engineering; Its description is seen the true tumor chemical experiment from book 3-carbohydrate I, gp (Tokyo Kagaku Dojin), the Japanese biological chemistry chief editor of association (1988); Cell engineering supplementary issue, experimental technique be from book, carbohydrate biological experimental method, gp, glycolipid and protein-polysaccharide (ShujuN-sha), Naoyuki Taniguchi, Akemi Suzuki, Kiyoshi Furukawa and Kazuyuki Sugawara chief editor (1996); Molecular cloning, second edition; The modern molecular biology method; With similar document.In the method that biochemical method comprises, enzymic activity is measured through using a kind of enzyme spcificity substrate and analogue.Gene engineering comprises that Northern analyzes, the method for the mRNA amount of RT-PCR and the gene of similarly measuring codase.
The method of confirming the phytohemagglutinin resistant cell comprises definite GDP-Fucose synthetic enzyme, and α 1, the method for 6-Fucose modifying enzyme or GDP-Fucose transhipment expression of enzymes, the method for culturing cell in the substratum that directly adds phytohemagglutinin.α 1 in the cell particularly, the α 1 of one of 6-Fucose modifying enzyme, 6-fucosyltransferase mRNA expression amount.Wherein α 1, and the cell that 6-Fucose modification enzyme activity reduces has resistance to phytohemagglutinin.
And; With α 1,6-Fucose modifier is active to be reduced the morphological change that causes and selects the method for cell for the basis, comprises that the sugar chain structure with the antibody molecule of preparation is the method that transformant is selected on the basis; Use the method for the sugar chain structure selection transformant of cytolemma gp, and similar approach.Use the sugar chain structure that produces antibody molecule to select the method for transformant to be included in the method described in following the 5th.Use the sugar chain structure of cytolemma gp to select the method for transformant to comprise that selection has the clone of resistance to the phytohemagglutinin that can discern sugar chain structure, wherein α-key combination is passed through in the 6-position that is connected the N-acetylglucosamine in the sugar chain complex body reduction end with the N-glucosides, the 1-position of Fucose.Example comprises the method for using phytohemagglutinin, and Somatic Cell.Mol.Gene (gene) t. is seen in its description, 12, 51 (1986).
As lectin, can use any lectin, as long as it is to discern 1 of Fucose is incorporated into the compound sugar chain reducing end under neutral of N-glucosidic linkage through the α key the lectin of sugar chain of 6 of N-acetyl-glucosamines.Example comprises LcA LCA (deriving from the LCA of Lens culinaris); Pisum sativum agglutinin PSA (deriving from the pisum sativum agglutinin of Pisum sativum); A kind of broad bean lectin VFA (deriving from the lectin of Vicia faba), dried orange peel auricularia auriculajudae lectin AAL (deriving from the lectin of Aleuria aurantia) or the like.
Specifically the selection of host cell of the present invention can be through culturing cell 1 day to 2 weeks; Preferred 3 days to 1 week; Containing concentration is tens μ g/ml to 10mg/ml, cultivates in the substratum that preferred 0.5 to 2.0mg/ml phytohemagglutinin is formed, the go down to posterity cell of survival or the clone who picks out; Be transferred in the culturing bottle, in containing the substratum of phytohemagglutinin, cultivate with continued.
(b) prepare cell of the present invention through the RDO method
The preparation of cell of the present invention can be passed through the RDO method, target coding for alpha 1, and the gene of 6-Fucose modifying enzyme for example carries out as follows.
Preparation coding for alpha 1, the cDNA or the genomic dna of 6-Fucose modifying enzyme.
Measure the cDNA of preparation or the nucleotide sequence of genomic dna.
Dna sequence dna to confirm is the basis, design and the synthetic a part of translation area that contains goal gene, the RDO construction of the suitable length of a part of non-translational region or a part of intron.
The acquisition of cell of the present invention can be selected transformant then through synthetic RDO is imported in the host cell, in transformant, and the target enzyme, just α 1, in the 6-Fucose modifying enzyme sudden change taken place.
As host cell, can use any cell such as yeast, zooblast, insect cell or vegetable cell are as long as it contains coding for alpha 1, the gene of 6-Fucose modifying enzyme.Example is included in the host cell described in following the 3rd.
The method that RDO is imported in the multiple host cell is included in the recombinant vectors that the multiple host cell of importing described in following the 3rd is fit to.
Preparation coding for alpha 1, the method for the cDNA of 6-Fucose modifying enzyme is included in " preparation method of cDNA " and the similar approach described in 1 (1).
Preparation coding for alpha 1, the method for the genomic dna of 6-Fucose modifying enzyme are included in 1 (1) " preparation method of genomic dna " and similar approach described in (a).
The mensuration of the nucleotide sequence of DNA can be through digesting with suitable Restriction Enzyme; Dna fragmentation is cloned among plasmid such as the pBluescript SK (-) (StrataGene (gene) manufacturing); The clone is reacted; This reaction is used as the method for analysis of nucleotide sequences usually, like people's such as Sanger dideoxy method [Proc.Natl.Acad Sci.USA 74, 5463 (1977)] or similar approach, through automatic nucleotide sequence analysis appearance, analyze the clone then like A.L.F.DNA sequenator (Pharmacia manufacturing) or similar approach.
RDO can prepare through the method or the employing dna synthesizer of routine.
Select wherein through RDO is imported in the host cell; At the target enzyme, coding for alpha 1, the method for the cell that having in the gene of 6-Fucose modifying enzyme suddenlys change takes place; Comprise the method for suddenling change in the direct detection genome; Molecular cloning is seen in its description, second edition, modern molecular biology method and similar approach.
And, also can use 1 (1) described in (a) with α 1,6-Fucose modification enzyme activity is changed to " the selecting the method for transformant " on basis.
The RDO construction can be according to Science (science), 2731386 (1996), Nature Medicine (natural drug), 4, 285 (1998), Hepatology (hepatology), 25, 1462 (1997), Gene Therapy (gene therapy), 5, 1960 (1999); J Mol.Med. (molecular medicine magazine)., 75, 829 (1997), Proc.Natl.Acad.Sci. (institute of AAS newspaper) USA, 96, 8774 (1999), Proc.Natl.Acad.Sci. (institute of AAS newspaper) USA, 96, 8768 (1999), Nuc.Acids Res (nucleic acids research)., 27, 1323 (1999), Invest.Dematol., 111,1172 (1998), Nature Biotech. (Nature Biotechnol), 16, 1343 (1998); Nature Biotech. (Nature Biotechnol), 18, 43 (2000); Nature Biotech. (Nature Biotechnol), 18, the method design of describing in 555 (2000) etc.
(c) prepare cell of the present invention through the method for using transposon
Cell of the present invention can use a kind of transposon system induction sudden change to prepare, and Nature Genet. (natural genetics) is seen in its description, 25, 35 (2000) or similar document, then with α 1, the activity of 6-Fucose modifying enzyme, or the sugar chain structure of the antibody molecule of preparation or cytolemma gp be a basis selection two mutants.
The transposon system is through in karyomit(e), inserting the system that foreign gene brings out sudden change at random; The foreign gene that wherein is inserted between the transposon generally is used as the carrier that brings out sudden change, and in cell, imports the translocase expression vector that is used for inserting at random at karyomit(e) gene simultaneously.
Can use any translocase, as long as the sequence of its fit for service transposon.
As foreign gene, can use any gene, as long as it can bring out sudden change in the DNA of host cell.
As cell, can use any cell such as yeast, zooblast, insect cell or vegetable cell are as long as it contains coding target α 1, the gene of 6-Fucose modifying enzyme.Example is included in the host cell described in following the 3rd.
For the gene of encoding antibody molecule is imported in the cell of the present invention as host cell, can use the method for the recombinant vectors that the multiple host cell of importing described in following the 3rd is fit to.
With α 1,6-Fucose modification enzyme activity is the basis, select the method for two mutants be included in 1 (1) described in (a) with α 1,6-Fucose modification enzyme activity is changed to " the selecting the method for transformant " on basis.
(d) prepare cell of the present invention through antisense method or rnase method
Cell of the present invention prepares through antisense method or Yeast Nucleic Acid enzyme process, and Cell Technology is seen in the description of method, 12, 239 (1993); BIO/TECHNOLOGY, 17, 1097 (1999); Hum.Mol Genet., 5, 1083 (1995); Cell Technology, 13, 255 (1994); Proc.Nail Acad.Sci.USA, 96, 1886 (1999); Or similar document, as passing through target coding for alpha 1, the gene of 6-Fucose modifying enzyme in the following manner.
The cDNA or the genomic dna of preparation goal gene.
Measure the cDNA of preparation or the nucleotide sequence of genomic dna.
Dna sequence dna to confirm is the basis, and design contains a part of translation area, the inverted defined gene of the suitable length of a part of non-translational region or a part of intron or the rnase construction of goal gene.
For antisence gene or rnase in cell, through the fragment of the DNA for preparing or the downstream that total length is inserted into suitable expression vector promotor are prepared recombinant vectors.
Obtain transformant through recombinant vectors being imported the host cell that is fit to expression vector.
Cell of the present invention can be the basis through the activity with goal gene encoded protein matter, selects transformant and obtains.Cell of the present invention also can be basis through the sugar chain structure with the sugar chain structure of cytolemma gp or preparation antibody molecule, selection transformant and obtaining.
As the host cell that is used to produce cell of the present invention, can use any cell, like yeast, zooblast, insect cell or vegetable cell are as long as it contains goal gene.Example is included in the host cell described in following the 3rd.
As expression vector, use can be in host cell self-replicating, maybe can be integrated into the carrier in the karyomit(e), the promotor place that it contains has been transmitted the inverted defined gene or the rnase of design.Example is included in the expression vector described in following the 3rd.
As the method that gene is imported in the multiple host cell, can use the method for the recombinant vectors that the multiple host cell of importing described in the 3rd below is fit to.
The method that obtains goal gene cDNA or genomic dna be included in 1 (1) (a) in method described in " preparation method of cDNA " and " preparation method of genomic dna ".
Activity with goal gene encoded protein matter is the basis, system of selection be included in 1 (1) (a) in method described in " selecting the method for transformant ".
And; Reduce caused morphological change with the activity of goal gene encoded protein matter and be the basis; The method of selection cell comprises that the sugar chain structure with the antibody molecule of preparation is the method that transformant is selected on the basis; Sugar chain structure with cytolemma gp is the method that transformant is selected on the basis, and similar approach.The sugar chain structure of antibody molecule with preparation serves as that the basis selects the method for transformant to comprise the method described in following the 5th.With the sugar chain structure of cytolemma gp serves as that the basis selects the method for transformant to comprise that above-mentioned selection has the method for the bacterial strain of resistance to the phytohemagglutinin that can discern sugar chain structure, and α-key combination is passed through in the 6-position that the 1-position of Fucose is connected the N-acetylglucosamine in the sugar chain complex body reduction end in this sugar chain structure with the N-glucosides.Example comprises the method for using phytohemagglutinin, and Somatic Cell.Mol.Gene (gene) t., 12,51 (1986) are seen in its description.
And cell of the present invention can not use expression vector and obtains, and through directly antisense oligonucleotide or rnase being imported in the host cell, they are to classify basic design as with the nucleotides sequence of goal gene.
Antisense oligonucleotide or rnase can or use dna synthesizer to prepare through usual method.In particular; It prepares available continuous 5 to 150 bases that have; Preferred 5 to 60 bases, more preferably the sequence information of the oligonucleotide of 10 to 40 base corresponding sequence is the basis, in the nucleotide sequence of goal gene cDNA and genomic dna; Carry out through synthetic oligonucleotide, this oligonucleotide is corresponding to oligonucleotide complementary sequence (antisense oligonucleotide) or contain the rnase of oligonucleotide sequence.
Oligonucleotide comprises the verivate (after this being called " oligonucleotide verivate ") of oligo rna and oligonucleotide.
The oligonucleotide verivate comprises that the phosphodiester bond in the oligonucleotide wherein is converted into the oligonucleotide verivate of thiophosphoric acid key; Wherein the phosphodiester bond in the oligonucleotide is changed into the oligonucleotide verivate of N3 '-P5 ' phosphoamidate key; Wherein ribose in the oligonucleotide and phosphodiester bond are changed into the oligonucleotide verivate of peptide-nucleic acid key; Wherein the uridylic in the oligonucleotide is by the oligonucleotide verivate of C-5 propynyluracil replacement; Wherein the uridylic in the oligonucleotide is by the oligonucleotide verivate of C-5 thiazole uridylic replacement; Wherein the cytosine(Cyt) in the oligonucleotide is by the oligonucleotide verivate of C-5 propynylcytosine replacement; The oligonucleotide verivate of the cytosine(Cyt) replacement of being modified by azophenlyene of the cytosine(Cyt) in the oligonucleotide wherein, wherein the ribose in the oligonucleotide by 2 '-the oligonucleotide verivate of O-propyl group ribose replacement, wherein the ribose in the oligonucleotide by 2 '-oligonucleotide verivate [the Cell Technology (Saibo Kogakn) of methoxyethoxy ribose replacement; 16,1463 (1997)].
(e) prepare cell of the present invention through the RNAi method
Cell of the present invention can be invented proteic gene through RNAi (RNA interference) method target code book and prepare, and is for example following.The RNAi method is meant that double-stranded RNA is imported in the cell, is present in the cell mRNA with the RNA sequence homology and is decomposed and destroys the method for inhibition of gene expression.
The cDNA of preparation goal gene.
Measure the nucleotide sequence of the cDNA of preparation.
Dna sequence dna to confirm is the basis, designs the RNAi gene constructs of the suitable length of being made up of a part of translation area or the non-translational region of goal gene.
For expressed rna i gene in cell, through the total length of the DNA for preparing or the downstream that fragment is inserted into suitable expression vector promotor are prepared recombinant vectors.
Obtain transformant through recombinant vectors being imported the host cell that is fit to expression vector.
Cell of the present invention can goal gene encoded protein matter antibody molecule or the sugar chain structure of cytolemma gp of active or preparation be the basis, obtain through the selection transformant.
As host cell, can use any cell, like yeast, zooblast, insect cell or vegetable cell are as long as the antibody molecule that it contains the coding preparation is the gene of target.Example is included in the host cell described in following the 3rd.
As expression vector, use can be in host cell self-replicating, maybe can be integrated into the carrier in the karyomit(e), the promotor place that it contains has been changed over to the RNAi gene of design.Example is included in the expression vector described in following the 3rd.
As the method that gene is imported in the multiple host cell, can use the method for the recombinant vectors that the multiple host cell of importing described in following the 3rd is fit to.
With the activity of goal gene encoded protein matter serves as that the basis is selected the method for transformant or served as that the basis selects the method for transformant to be included in 1 (1) method described in (a) with cytolemma glycoprotein candy chain structure.Antibody molecule sugar chain structure with preparation serves as that the basis selects the method for transformant to be included in the method described in following the 5th.
The method of the cDNA of preparation goal gene coded protein be included in 1 (1) (a) in method and similar approach described in " preparation method of cDNA ".
And cell of the present invention can not use expression vector and obtains, and can classify the RNAi gene of basic design through direct importing as with the nucleotides sequence of goal gene.
Can or use dna synthesizer to prepare the RNAi gene through ordinary method.
The design of RNAi gene constructs can be seen Nature (nature) according to following described method, 391, 806 (1998); Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 95, 15502 (1998); Nature (nature), 395, 854 (1998); Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 96, 5049 (1999); Cell (cell), 95, 1017 (1998); Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 96,1451 (1999); Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 95, 13959 (1998); Nature (nature) Cell Biol, 2, 70 (2000); With similar document.
The RNA that in RNAi method of the present invention, uses comprises the corresponding RNA of DNA of relevant sugar chain modified zymoprotein with coding; The 6-position that the 1-position of Fucose is connected the N-acetylglucosamine in the sugar chain complex body reduction end in the described sugar chain with the N-glucosides is through α-key combination; And preferred and coding for alpha 1; The RNA that the DNA of 6-fucosyltransferase is corresponding; This enzyme is as relevant sugar chain modified zymoprotein, and the 6-position that the 1-position of Fucose is connected the N-acetylglucosamine in the sugar chain complex body reduction end in the described sugar chain with the N-glucosides is through α-key combination.
RNA as using in the RNAi method of the present invention can use any RNA, as long as the double-stranded RNA that it is made up of RNA and complementary RNA thereof can reduce α 1, the amount of 6-fucosyltransferase mRNA.About the length of RNA, this RNA is preferred 1 to 30, more preferably 5 to 29, still more preferably 10 to 29 and 15 to 29 continuous RNA most preferably.
(2) prepare cell of the present invention through the method that imports dominant negative mutant
Cell of the present invention can be through use importing the method target α 1 of proteinic dominant negative mutant, 6-Fucose modifying enzyme and preparing.
As dominant negative mutant, example is for relating to intracellular sugar nucleotide, and the GDP-Fucose is transported to the protein of golgi body.
Transhipment of known intracellular sugar nucleotide on the film of endoplasmic reticulum or golgi body be with dimeric form acting [J.Biol.Chem. (journal of biological chemistry), 275, 17718 (2000)].Report is also arranged; When the two mutants of intracellular sugar nucleotide transhipment during forced expression, forms heterodimer with wild-type transhipment in cell, the heterodimer of formation has the activity [J.Biol.Chem. (journal of biological chemistry) that suppresses the wild-type homodimer; 275,17718 (2000)].Therefore, the two mutants of preparation intracellular sugar nucleotide transhipment, and in the transfered cell so that work as dominant negative mutant.Can adopt site-directed mutagenesis to prepare two mutants, molecular cloning is seen in its description, second edition, modern molecular biology method and similar document.
Cell of the present invention can adopt the α 1 for preparing according to the method described in the following document in the above, and the dominant negative mutant gene of 6-Fucose modifying enzyme prepares, and sees molecular cloning; Second edition, modern molecular biology method, operation mouse embryo; Or similar document, for example following.
Preparation α 1, the dominant negative mutant gene of 6-Fucose modifying enzyme.
The full length DNA of dominant negative mutant gene with preparation is basis, if desired, prepares the dna fragmentation of the suitable length that contains the part coded protein.
Promotor downstream through dna fragmentation or full length DNA being inserted into suitable expression vector prepare recombinant vectors.
Through being imported in the host cell that is fit to expression vector, recombinant vectors obtains transformant.
Host cell of the present invention can α 1, and the sugar chain structure of active or the preparation antibody molecule or the cytolemma gp of 6-Fucose modifying enzyme is selected transformant for the basis and prepared.
As host cell, can use any cell, like yeast, zooblast, insect cell or vegetable cell are as long as it contains the gene of the target protein of encoding.Example is included in the host cell of describing in following the 3rd.
As expression vector, use can be in host cell self-replicating, maybe can be integrated into the carrier in the karyomit(e), the transcribing of DNA of the promotor place coding purpose dominant negative mutant that it contains can be influenced.Example is included in the expression vector described in following the 3rd.
As the method that gene is imported in the multiple host cell, can use the method for the recombinant vectors that the multiple host cell of importing described in the 3rd below is fit to.
With the activity of target protein serves as that the basis is selected the method for two mutants or served as that the basis selects the method for two mutants to comprise the method described in top 1 (1) with the sugar chain structure of cytolemma gp.The sugar chain structure of antibody molecule with preparation serves as that the basis selects the method for two mutants to be included in the method described in following the 4th.
(3) method in the enzyme that imports of will suddenling change
The preparation of cell of the present invention can import coding for alpha 1 through suddenling change, and in the gene of 6-Fucose modifying enzyme, selects the purpose clone who wherein in enzyme, undergos mutation then.
Relevant sugar chain modified enzyme comprises α 1; The 6-fucosyltransferase; Alpha-L-fucosidase and analogue, the 6-position that the 1-position of Fucose is connected the N-acetylglucosamine in the sugar chain complex body reduction end in the described sugar chain with the N-glucosides is through α-key combination.
Method comprises 1) a kind of method; Wherein required clone is selected from and handles parental cell with mutagenic compound is the two mutants that produces of induced mutation or with α 1; The activity of 6-Fucose modifying enzyme is the two mutants of basic spontaneous generation; 2) a kind of method; Wherein required clone is selected from that to use mutagenic compound to handle parental cell be the two mutants that produces of induced mutation or is the two mutants and 3 of basic spontaneous generation with the sugar chain structure of the antibody molecule of preparation) a kind of method, wherein required clone is selected from that to use mutagenic compound to handle parental cell be the two mutants that produces of induced mutation or is the two mutants of basic spontaneous generation with the sugar chain structure of cytolemma gp.
As the processing of induced mutation, can use any treatment process, as long as it can induce the DNA point mutation in the parental cell system, deletion or phase shift mutation.Example comprises uses ethylnitrosourea, nitrosoguanidine, and benzopyrene or acridine pigment are handled, and handle with radioactive rays.Multiple alkylating agent and carcinogen also can be used as mutagenic compound.The method that makes mutagenic compound act on cell is included in tissue culture technique, the third edition (Asakura Shoten), and the Japanese tissue culture chief editor of association (1996), Nature (nature) Genet., 24, 314 (2000) and similar document in the method described.
The two mutants of spontaneous generation is included in not to be used special sudden change and induces processing under the common cell culture condition, cultivate the two mutants of spontaneous formation through continuous passage.
With α 1; The activity of 6-Fucose modifying enzyme is the method that the purpose clone is selected on the basis; With the sugar chain structure of antibody molecule of preparation serve as the basis select the purpose sugar chain method and with the sugar chain structure of cytolemma gp serve as the basis select purpose clone's method be included in 1 (1) described in (a) with α 1,6-Fucose modification enzyme activity is changed to " the selecting the method for transformant " on basis.
(4) method of transcribing or translating of inhibition GDP-Fucose translocator
Host cell of the present invention can pass through target coding for alpha 1, the gene of 6-Fucose modifying enzyme with adopt sense-rna/dna technique suppress the transcribing or translating of goal gene [BioScience (science) and Industry, 50, 322 (1992); Chemistry (Kagaku), 46, 681 (1991); Biotechnology, 9, 358 (1992); Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell Technology, 16, 1463 (1997)], three-herix technology [Trends in Biotechnology, 10, 132 (1992)] or similar approach prepare.
2. prepare transgenic nonhuman animal of the present invention or plant or its filial generation
Transgenic nonhuman animal of the present invention or plant or its filial generation are the adorned transgenic nonhuman animal of its genomic gene or plant or its filial generation; But the mode of modifying is the sugar chain modified enzymic activity Be Controlled of relevant antibody molecule; Can be according to the method described in the 1st from embryonic stem cell; Fertilized egg cell or vegetable cell, through target coding for alpha 1, the gene of 6-Fucose modifying enzyme and preparing.
Relevant sugar chain modified enzyme comprises α 1; The 6-fucosyltransferase; Alpha-L-fucosidase and analogue, the 6-position that the 1-position of Fucose is connected the N-acetylglucosamine in the sugar chain complex body reduction end in the described sugar chain with the N-glucosides is through α-key combination.
Special method is described below.
In transgenic nonhuman animal; The preparation of embryonic stem cell of the present invention can be through being applied to the embryonic stem cell of required non-human animal such as ox, sheep, goat, pig, horse, mouse, rat, poultry, monkey, rabbit or similar animal in the method described in the 1st; Wherein α 1, but the activity Be Controlled of 6-Fucose modifying enzyme.
In particular, the clone of sudden change can be produced through using a kind of known homologous recombination technique, and wherein coding for alpha 1, the gene of 6-Fucose modifying enzyme be inactivated or with any sequence replace [like, Nature (nature), 326,6110,295 (1987); Cell (cell), 51., 3,503 (1987); Or similar document].Use the mutant clon for preparing, the preparation that contains embryonic stem cell clone and Normocellular chimeric individuality can get into the method for fertilised non-human eggs blastaea or pass through the mosaic aggregation method through the injection mosaic.The hybridization of chimeric individuality and normal individual, so that obtain transgenic nonhuman animal, α 1 in the whole machine body cell wherein, the activity of 6-Fucose modifying enzyme reduces.
According at gene target, hands-on approach, IRL Press at Oxford University Press (1993); Method described in the Biomanual book series 8, gene target, use ES cell preparation mutant mice, Yodo-sha (1995) or similar document is that the homologous recombination of goal gene prepares targeting vector.Targeting vector can be used substituted type, any in insert type and the gene trap type.
As the method that targeting vector is imported in the embryonic stem cell, can use any method, as long as it can import DNA in the zooblast.Example comprise electroporation [Cytotechnology (electroporation), 3, 133 (1990)], calcium phosphate method (No. 227075/90, the not unexamined patent application that Japan has announced); Fat dyes method [Proc.Nail Acad Sci.USA, 84,7413 (1987)]; Injection [operation mouse embryo, laboratory manual], method (particle gun) (No. 2606856, the Japanese Patent of use particle gun; No. 2517813, Japanese Patent), DEAE-VISOSE method [Biomanual book series 4-gene transmits and expression analysis (Yodo-sha), Takashi Yokota and Kenichi Arai chief editor (1994)]; Virus vector method [operation mouse embryo, second edition] and similar approach.
Effectively select the method for homologous recombination body to comprise following method, as just selecting, promotor is selected, negative select or polyA selects, and gene target is seen in its description, hands-on approach, IRL Press at Oxford University Press (1993), or similar document.Particularly under the situation of the targeting vector that contains the hprt gene; It can be imported in the embryonic stem cell of hprt genetic flaw; Embryonic stem cell is cultivated and is being contained aminopterin; In the substratum of xanthoglobulin and thymus pyrimidine, carry out to select the just selection of hprt dna homolog recombinant chou through the homologous recombination body of selecting to contain the aminopterin resistance clone.Under the situation of the targeting vector that contains neomycin resistance gene, the embryonic stem cell that imports carrier is cultivated in containing the substratum of G418, just selects through the homologous recombination body of selecting to contain neomycin resistance gene.Containing under the targeting vector situation of DT gene; Cultivate the embryonic stem cell that imports carrier; Through selecting the growth clone not have negative selection (because the DT genetic expression of DT dna homolog recombinant chou; Be incorporated into simultaneously in the karyomit(e), by at random rather than homologous recombination import in the karyomit(e) recombinant chou because the toxicity of DT and can not grow).The method of selection purpose homologous recombination body comprises the Southern hybridization (molecular cloning, second edition) of genomic dna, PCR [PCR method, Academic Press (1990)] and similar approach in the clone who has selected.
When embryonic stem cell adopts the mosaic aggregation method to be imported in the zygote, preferred usually budding zygote of using before 8 cell stages.When embryonic stem cell uses injection mosaic method to be imported in the zygote, preferred usually budding zygote of using between 8 cell stages to blastula stage.
When zygote is transplanted in the female mice, the zygote that preferred artificial graft or plantation obtain from the false pregnancy female mice, its fertilization be through with brought out by the male non-human mammal mating of ligation.Although the female mice of false pregnancy can obtain through the mating of nature, after using luteinising hormone-releasing hormo (after this being called " LHRH ") or its analogue, the false pregnancy female mice that is brought out fertilization can be through obtaining with the male mice mating.The analogue of LHRH comprises [3,5-Dil-Tyr5]-LHRH, [Gln8]-LHRH, [D-Ala6]-LHRH, des-Gly1O-[D-His (Bzl) 6]-LHRH ethamine and analogue.
Fertilized egg cell's of the present invention preparation also can be through being used for the method described in the 1st zygote of purpose non-human animal such as ox, sheep, goat, pig, horse, mouse, rat, poultry, monkey, rabbit or similar animal; α 1 in the said zygote, and the activity of 6-Fucose modifying enzyme reduces.
Wherein α 1; The preparation of the transgenic nonhuman animal that 6-Fucose modification enzyme activity reduces can migrate in the false pregnancy female uterine tube or uterus through the fertilized egg cell with preparation; Use is at operation mouse embryo, the embryo transfer method described in second edition or the similar document, animal childbirth then.
In transgenic plant, wherein α 1, and the callosal preparation of the present invention that 6-Fucose modification enzyme activity reduces can be through being applied to the method described in the 1st in the corpus callosum or cell of purpose plant.
Wherein α 1, and the preparation of the transgenic plant that 6-Fucose modification enzyme activity reduces can be according to known method, through the corpus callosum of using the culture medium culturing formed by growth hormone and phytokinin to prepare, make it and break up again [Tissue Culture, 20(1994); Tissue Culture, 21(1995); Trends in Biotechnology, 15, 45 (1997)].
3. produce the method for antibody compositions
The acquisition of antibody compositions can be expressed it through using the method described in the document below in host cell, see molecular cloning, second edition; The modern molecular biology method, antibody, laboratory manual; Cold Spring Harbor Laboratory, 1988 (after this being called " antibody "); Monoclonal antibody: principle and practice, the third edition, Acad.Press, 1993 (after this being sometimes referred to as " monoclonal antibody "); And antibody engineering, hands-on approach, IRL Press at Oxford University Press (after this being sometimes referred to as " antibody engineering "), for example following.
The cDNA of preparation antibody molecule.
The full-length cDNA of antibody molecule with preparation is basis, if desired, prepares the dna fragmentation of the suitable length of the composition that contains the part coded protein.
Downstream through dna fragmentation or full-length cDNA being inserted into suitable expression vector promotor prepare recombinant vectors.
The transformant that produces antibody molecule can obtain through recombinant vectors is imported in the host cell that is fit to expression vector.
As host cell, can use any yeast, zooblast, insect cell, vegetable cell or analogue, as long as it expresses goal gene.
Relate to and be connected sugar chain modified enzyme with the Fc district bonded N-glucosides of antibody molecule and also can be used as host cell through cell such as yeast, zooblast, insect cell, vegetable cell or the analogue that gene engineering is imported into.
The host cell that is used for producing antibody compositions is included in top 1 prepared cell of the present invention.
As expression vector, use can be in host cell self-replicating, maybe can be integrated into the carrier in the karyomit(e), the promotor place that it contains can transmit the DNA of coding purpose antibody molecule.
According to 1 (1) (a) in method described in " preparation method of cDNA ", use, for example the special probe primer of purpose antibody molecule prepares cDNA from people or inhuman tissue or cell.
When yeast as host cell, expression vector comprises YEP 13 (ATCC 37115), YEp24 (ATCC 37051), YcpS50 (ATCC 37419) etc.
Can use any promotor, as long as it can play a role in yeast.Example comprises the promotor of the gene such as the HK gene of glycolytic pathway, PHO5 promotor, PGK promotor, GAP promotor, ADH promotor, gal 1 promotor, gal 10 promotors, heat shock protein(HSP) promotor, MF α 1 promotor, CUP 1 promotor etc.
Host cell comprises the kind that belongs to following, like yeast belong, and Schizosaccharomyces; Genus kluyveromyces, Trichosporon, Schwanniomyces belongs to and similar kind; Like yeast saccharomyces cerevisiae; Pombe Peng shellfish fission yeast, Kluyveromyces lactis, trichosporon pullulans and Schwanniomyces alluvins.
As the method that imports recombinant vectors, can use any method, as long as it can import DNA in the yeast.Example comprise electroporation [Methods in Enzymology, 194,182 (1990)], the spheroplast method [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 84, 1929 (1978)], the Lithium Acetate method [J.Bacterial, 153,163 (1983)], at Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 75, 1929 (1978) middle method and the similar approach of describing.
When zooblast when the host cell, expression vector comprises pcDNAI, pcDMS (obtaining) from Funakoshi, [Japan has announced No. 22979/91, the patented claim of not substantive examination to pAGE107; Cytotechnology (electroporation), 3, 133 (1990)], pAS3-3 (Japan has announced No. 227075/90, the patented claim of not substantive examination), pCDMS [Nature (nature), 329,840 (1987)], pcDNAI/Amp (Invitrogen manufacturing), pREP4 (Invitrogen manufacturing), pAGE 103 [J.Biochemistry (biological chemistry), 101,1307 (1987)], pAGE210 and analogue.
Can use any promotor, as long as it can play a role in zooblast.Example comprises IE (extremely early stage) promotor of cytomegalovirus (CMV) gene, the early promoter of SV40, retroviral promotor, the promotor of rhMT, heat-shocked promotor, SR α promotor etc.The enhanser of people CMVIE gene can use with promotor.
Host cell comprises people's cell such as Namalwa cell; MC such as COS cell, Chinese hamster cell such as Chinese hamster ovary celI or HBT5637 (Japan has announced the patented claim No.299/88 of not substantive examination), rat myeloma cell; Murine myeloma cell; From the cell of Syria hamster kidney, embryonic stem cell, fertilized egg cell and similar cell.
As the method that imports recombinant vectors, can use any method, as long as it can import DNA in the zooblast.Example comprise electroporation [Cytotechnology (electroporation), 3, 133 (1990)], calcium phosphate method (Japan announced the patented claim No.227075/90 of not substantive examination), fat dye method [Proc.Natl.Acad Sci.USA, 84, 7413 (1987)], injection [operation mouse embryo; Laboratory manual], the method (particle gun) (No. 2606856, Japanese Patent, No. 2517813, Japanese Patent) of employing particle gun; [Biomanita transmits and expression analysis (Yodo-sha) from book 4-gene DEAE-VISOSE method; Takashi Yokota and Kenichi Arai chief editor (1994)], virus vector method (operation mouse embryo, second edition) and analogue.
When insect cell as the host, protein can be through molecular biology method in modern times, rhabdovirus expression vector, laboratory manual; W.H.Freeman and Company, New York (1992), Bio/Technology; 6,47 (1988) or similar document described in method express
Protein can be expressed with the recombinant virus infection insect cell through obtaining recombinant virus in the supernatant of cultivating insect then.
The gene that in this method, uses imports carrier and comprises pVL1392, pVL1393, pBlueBacIII (all are all made by Invitrogen) and analogue.
Baculovirus comprises California clover noctuid nucleopolyhedrosis virus, can be by the insect infection of lopper worm family.
Insect cell comprises ovocyte [the modern molecular biology method of greedy noctuid Sf9 in meadow and Sf21; Rhabdovirus expression vector; Laboratory manual; W.H.Freeman and Company, New York (1992)], the ovocyte of trichoplusia ni High 5 (Invitrogen manufacturing) and similar cell.
Recombination imported the common method that imports of carrier and baculovirus comprise calcium phosphate method (Japan has announced No. 227075/90, the patented claim of not substantive examination) for preparing recombinant virus, fat dye method [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 84, 7413 (1987)] and similar approach.
When vegetable cell was used as host cell, expression vector comprised Ti-plasmids, tobacco mosaic disease poisonous carrier and similar substrates.
As promotor, can use any promotor, in vegetable cell as long as it can play a role.Example comprises cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promotor and similar promotor.
Host cell comprises tobacco, yam, Radix Dauci Sativae, soybean, rape, clover, rice, wheat, barley and similar vegetable cell.
As the method that imports recombinant vectors, can use any method, as long as it can import DNA in the vegetable cell.Example comprises and uses edaphic bacillus (Japan has announced No. 140885/84, the patented claim of not substantive examination; Japan has announced No. 70080/85, the patented claim of not substantive examination; WO 94/00977) method; Electroporation (Japan has announced No. 251887/85, the patented claim of not substantive examination) uses the method (No. 2606856, Japanese Patent, No. 2517813, Japanese Patent) and the similar approach of particle gun (particle gun).
As the method for expressing antibodies gene, the secretion of Fc district and other proteic fusion roteins etc. produces, and expresses except direct expression, can be according at molecular cloning, and the method for describing in second edition or other documents is carried out.
When gene by yeast, zooblast when insect cell or vegetable cell are expressed, has been imported in the cell and has related to sugar chain synthetic gene, can pass is imported into the antibody molecule that gene has added sugar or sugar chain.
The acquisition of antibody compositions can in substratum, to produce and the accumulation antibody molecule, be reclaimed from the substratum that obtains through in substratum, cultivating acquired transformant then.Adopt the method for culture medium culturing transformant to carry out according to the method that is generally used for cultivating host cell.
As carbon source.Can use those by the carbon source of organism.Example comprises glucide such as glucose, fructose, sucrose, contains their molasses, starch and starch hydrolyzates; Organic acid such as acetate and propionic acid; Alcohols such as ethanol and propyl alcohol; And analogue.
Nitrogenous source comprises ammonia; Mineral acid or organic acid ammonium salt such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; The compound that other are nitrogenous; Peptone; Meat extract; Yeast extract; Steeping water; Casein hydrolysate; Soybean meal (soybean meal); The soybean meal hydrolyzate; Multiple fermentation cell and hydrolyzate thereof; And analogue.
Inorganics comprises potassium primary phosphate, potassium hydrogenphosphate, trimagnesium phosphate, sal epsom, sodium-chlor, ferrous sulfate, copper sulfate, lime carbonate and analogue.
Usually in aerobic condition such as the jolting cultivation or the stir culture system of taking a breath under water, cultivate.Preferred 15 to 40 ℃ of the temperature of cultivating, incubation time is usually at 16 hours to 7 days.In cultivation, pH maintains 3.0 to 9.0.With mineral acid or organic acid, alkaline solution, urea, lime carbonate, ammonia or analogue adjustment pH.
If desired, in cultivation, can add microbiotic such as penbritin or tsiklomitsin to substratum.
When using the recombinant vectors microorganism transformed that obtains as promotor through the use inducible promoter to be cultivated, can in substratum, add inductor if desired.For example; When using the recombinant vectors microorganism transformed that obtains through the lac promotor to be cultivated; Can in substratum, add sec.-propyl-β-D-sulfenyl semi-lactosi pyranoside; When using the recombinant vectors microorganism transformed that obtains through the trp promotor to be cultivated, can in substratum, add indole acrylic acid.
When the transformant of using zooblast to obtain as host cell is cultivated, substratum comprise normally used RPMI 1640 substratum [AMA's monthly magazine, 199,519 (1967)], Eagle ' s MEM substratum [science, 122,501 (1952)], Dulbecco ' s modified MEM substratum [virusology, 8, 396 (1959)], 199 substratum [biomedical association progress, 73, 1 (1950)] and Whitten substratum [grow engineering experiment handbook-preparation transgenic mice (KodaN-sha), M.Katsuki edits (1987)], the substratum and the analogue of interpolation calf serum etc.
Cultivate usually at 6 to 8,30 to 40 ℃ of pH, 5%CO 2Under carried out 1 to 7 day.
If desired, can in cultivation, add microbiotic such as kantlex or penicillium mould.
Cultivate and use insect cell to comprise normally used TNM-FH substratum (Pharmingen manufacturing) as the substratum of the transformant of host's acquisition; Sf-900 II SFM substratum (Life Technologies manufacturing); ExCell 400 and ExCell 405 (all making) by JRH BioScience (science) s; Grace insect substratum [Nature (nature) 195,788 (1962)] and similar substratum.
Cultivate and carried out 1 to 5 day 6 to 7,25 to 30 ℃ of neutral pH usually.
In addition, if desired, in culturing process, can in substratum, add microbiotic such as qingfengmeisu qiong.
Use vegetable cell to can be used as cell cultures, or after its differentiation is become vegetable cell or organ, cultivate as the transformant that host cell obtains.The substratum of culture transformation body comprises normally used Murashige and Skoog (MS) substratum and White substratum, adds plant hormone such as growth hormone, the substratum of cytokine etc. and similar substratum.
Cultivate and carried out 3 to 60 days 5 to 9,20 to 40 ℃ of pH usually.
If desired, in cultivation, can in substratum, add microbiotic such as kantlex or Totomycin.
Therefore; The generation of antibody compositions can be through cultivating from yeast; The transformant of zooblast or vegetable cell according to common cultural method, thereby produces and the accumulation antibody compositions; From substratum, reclaim antibody compositions then, be inserted into the DNA of encoding antibody molecule in the recombinant vectors that wherein transformant contains.
The method that produces antibody compositions is included in the cell inner expression method in the host cell, from the cell exocrine method of host cell, the method that on the host cell membrane adventitia, produces.Can come system of selection according to the change of the antibody compositions structure of employed host cell or generation.
When antibody compositions of the present invention produces in host cell or on the host cell membrane adventitia, according to people's such as Paulson method [J.Biol.Chem. (journal of biological chemistry), 264,17619 (1989)], people's such as Lowe method [Proc.Natl.Acad Sci.USA, 86, 8227 (1989), Gene (gene) s Develop., 4, 1288 (1990)], announced method and the similar approach of describing in No. 823021/94, the patented claim of not substantive examination in No. 336963/93, patented claim and Japan that Japan has announced not substantive examination, it can be by active secretion to the extracellular.
Just; Through using recombinant gene; The DNA that in expression vector, inserts an encoding antibody molecule gets into the DNA that coding is fit to the signal peptide of expressed antibody molecule; Expression vector is imported in the host cell, expressed antibody molecule then, purpose antibody molecule can be from host cell on one's own initiative by secretion to the extracellular.
Also can use dihydrofolate reductase gene to increase its output according to the method for describing in No. 227075/90, the patented claim of having announced not substantive examination in Japan through using a kind of gene amplification system.
In addition, antibody compositions also can produce through animal individual (transgenic nonhuman animal) or the plant individual (transgenic plant) that use has imported gene, and its structure is the differentiation again through the animal or plant cell that has been imported into gene.
When transformant is animal individual or plant individual, the generation of antibody compositions can should produce and the accumulation antibody compositions by individuality through raising or cultivating according to a kind of usual method, from animal or plant individual, reclaims antibody compositions then.
Through the method for using animal individual to produce antibody compositions comprise the method that a kind of purpose antibody compositions produces in the animal that makes up according to a kind of currently known methods quiding gene [American Journal of Clinical Nutrition, 63, 627S (1996); Bio/Technology, 9, 830 (1991)].
Under the situation of animal individual, the generation of antibody compositions can be through raising genetically modified non-human animal, and this animal has been imported into the DNA of encoding antibody molecule, thereby in animal, produce and the accumulation antibody compositions, from animal, reclaims antibody compositions then.Produce in the animal and the position of accumulation compsn comprises the ovum of milk (Japan has announced No. 309192/88, the patented claim of not substantive examination) and animal.As the promotor of using in the case, can use any promotor, in animal as long as it can play a role.Preferred example comprises mammary gland cell specificity promoter such as casein promoter, β casein promoter, beta lactoglobulin promotor, whey acidic protein promotor and similar promotor.
Method through using plant individual to produce antibody compositions comprises a kind of method, and wherein the generation of antibody compositions is through cultivating a kind of transgenic plant, this plant through known method be imported into the encoding antibody molecule DNA [Tissue Culture, 20(1994); Tissue Culture, 21(1995); Trends in Biotechnology, 15, 45 (1997)], in plant, to produce and the accumulation antibody compositions, from plant, reclaim antibody compositions then.
About the purifying of the antibody compositions that produces through transformant, be imported into the gene of encoding antibody molecule in this transformant, for example when antibody compositions is expressed with dissolved state in cell; Cell centrifugation after the cultivation reclaims; Be suspended in the water damping fluid, use ultrasonic oscillator then, French press; Manton Gaulin homogenizer, dynomill or similar approach destroy and obtain cell-free extract.From the supernatant that obtains through centrifugal cell-free extract, can obtain the purified product of antibody compositions, through using common enzyme purification technology like solvent extraction; Saltout with the ammonium sulfate desalination etc.; Use organic solvent deposit; Use the anion-exchange chromatography of resin such as diethylaminoethyl-(DEAE)-sepharose or DIAION HPA-75 (Mitsubishi Chemical manufacturing); Use the Zeo-karb of resin such as S-Sepharose FF (Pharmacia manufacturing); Use resin such as butyl-sepharose, the HC of phenyl-sepharose; Use the gel-filtration of molecular sieve; Affinity chromatography; Chromatofocusing; Electrophoresis such as isoelectric focusing; With similar independent use or unite the method for use.
When antibody compositions also when forming that solution is not at cell inner expression, reclaim cell in an identical manner, destroy with centrifugal, the not solution of antibody compositions partly is recovered as deposition.The antibody that reclaims not solution adopts the protein denaturant dissolving.Through dilution or dialysis dissolved solution, antibody compositions is processed normal three-dimensional structure, obtain the purified product of antibody compositions then through identical separation purification method.
When antibody compositions was secreted to the extracellular, the antibody compositions or derivatives thereof reclaimed from culture supernatant liquid.That is,, from soluble part, pass through the purifying preparation that identical separation purification method obtains antibody compositions through obtaining soluble part like centrifugal technical finesse substratum.
Therefore the antibody compositions that obtains comprises antibody, antibody fragment, the fusion rotein of being made up of the antibody Fc district, and analogue.
As the example that obtains antibody compositions, the method that produces humanized antibody compsn and Fc fusion rotein details below, but the also available mode that is similar to this method of other antibody compositions obtains.
A. prepare the humanized antibody compsn
(1) makes up the carrier that humanized antibody is expressed
The carrier that humanized antibody is expressed is the animal cell expression carrier that has inserted coding human heavy chain of antibody and L chain C district gene, and its structure can pass through in the expression vector with the gene clone precession thing cell of every CH of coding human antibody and CL.
The C district of people's antibody can be the CH and the CL of anyone antibody.Example comprises the C district (after this being called " hC γ 1 ") that belongs to the IgG1 hypotype in people's heavy chain of antibody, belongs to the C district (after this being called " hC κ ") of K type in people's light chain of antibody, and analogue.
As the gene of coding human antibody CH and CL, can use by exon with include molecular chromosomal DNA, also can use cDNA.
As the expression vector of zooblast, can use any carrier, as long as the gene of coding human Antibody C region can be inserted into wherein and expression therein.Example comprise pAGE 107 [Cytotechnology (electroporation), 3, 133 (1990)], pAGE103 [J.Biocfiem., 101, 1307 (1987)], pHSG274 [Gene (gene), 27, 223 (1984)], pKCR [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 78, 1527 (1981), pSG1 β d2-4 [Cytotechnology (electroporation), 4, 173 (1990)] etc.Promotor in the expression vector of zooblast and enhanser contain SV40 early promoter and enhanser [J.Biochem. (biological chemistry impurity), 101,1307 (1987)], Moroni murine leukemia virus LTR promotor [Biochem.Biophys.Res.Comnun., 149, 960 (1987)], IgH chain promotor [Cell (cell), 41, 479 (1985)] and enhanser [Cell (cell), 33, 717 (1983)] and analogue.
The humanized antibody expression vector that has made up can be used to expressing human chimeric antibody and people CDR-grafted antibody in zooblast.
(2) preparation method of coding nonhuman animal antibody V district cDNA
The cDNA of coding nonhuman animal antibody such as murine antibody VH and VL can following mode obtain.
The mRNA that cDNA extracts from the hybridoma that produces the purpose murine antibody is synthetic.Synthetic cDNA is cloned in carrier such as phage or the plasmid to obtain the cDNA library.Each recombinant phage or recombinant phage or recombinant plasmid of recombinant plasmid and the cDNA that contains the VL that encodes that contains the cDNA of the VH that encodes separates from the library, and the C district part or the V district that use existing murine antibody are partly as probe.Be determined at the complete nucleotide sequence of purpose murine antibody VH and VL on recombinant phage or the recombinant plasmid, the total length of extrapolating VH and VL from nucleotide sequence is by the sequence of calculation.
As the non-human animal, can use any animal such as mouse, rat, hamster or rabbit are as long as can therefrom produce hybridoma.
From hybridoma the preparation total RNA method comprise guanidine thiocyanate-trifluoracetic acid caesium method [Methods in Enzymology, 154, 3 (1987)] and similar approach, the method that from total RNA, prepares mRNA comprises plain post method [molecular cloning, laboratory manual, second edition, Cold Spring Harbor Laboratory Press (1989)] of oligomerization (dT)-anchoring fiber and similar approach.In addition, the embodiment of the test kit of preparation mRNA comprises Fast Track mRNA separating kit (Invitrogen manufacturing), Quick Prep mRNA purification kit (Pharmacia manufacturing) and analogue from hybridoma.
Synthetic cDNA comprises conventional method [molecular cloning with the method for preparing the cDNA library; Laboratory manual, second edition, Cold Spring Harbor Laboratory Press (1989); The modern molecular biology method; Supplementary issue 1-34], the method for use commercial reagent box, test kit such as Superscript TM, cDNA synthesizes pUC pUC (GEBCO BRL manufacturing) or the ZAP-cDNA synthetic agent box (Stratagene manufacturing) with plasmid clone, and analogue.
In the process of preparation cDNA library, having inserted the mRNA that from hybridoma, extracts can be any carrier as the carrier of template synthetic cDNA, as long as can insert cDNA.Embodiment comprise ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], XzapII (Stratagene manufacturing), λ gt10 and λ gt11 [dna clone, hands-on approach, I, 49 (1985)], LambdaBlueMid (Clontech manufacturing), λ ExCell, pT7T3 18U (Pharmacia manufacturing), pcD2 [Mol.Cell.Biol., 3, 280 (1983)], pUCIS [Gene, 33,103 (1985)] and analogue.
As being imported into from the intestinal bacteria in the cDNA library of phage or plamid vector construction, can use any intestinal bacteria, as long as can be imported into, express and reservation cDNA library.Embodiment comprise XL 1-Blue MRF [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088 and Y1090 [Science, 222, 778(1983)], NM522 [J.Mol, Biol., 166,1 (1983)], K802 [J.Mol.Biol., 16,118 (1966)], JM105 [Gene, 38,275 (1985)] and analogue.
Method as the cDNA clone who from the cDNA library, selects coding nonhuman animal antibody H chain and L chain V district; Can use the colony hybridization or the plaque hybridization [molecular cloning that adopt isotropic substance or fluorescence labeling probe; Laboratory manual; Second edition, Cold Spring Harbor Laboratory Press (1989)].The cDNA of coding VH and VL can be through preparing primer, carries out the polymerase chain reaction and prepare and [after this be called " PCR "; Molecular cloning, laboratory manual, second edition, Cold Spring Harbor Laboratory Press (1989); Modern molecular biology method, supplementary issue 1-34] use from mRNA or cDNA library synthetic cDNA as template.
The mensuration of cDNA nucleotide sequence can be through the cDNA that has selected with suitable Restriction Enzyme digestion; Dna fragmentation is cloned in the plasmid like pBluescript SK (-) (Stratagene manufacturing); Carry out people's such as normally used nucleotide sequence analysis method such as Sanger dideoxy method [Proc.Nat/.Acad.Sci.USA 74, 5463 (1977)] or the reaction of similar approach, automatic nucleotide sequence analysis appearance such as A.L.F.DNA sequenator (Pharmacia manufacturing) or analogous instrument analysis clone used then.
No matter antibody VH and VL full length amino acid sequence that whether acquired cDNA encodes and contain secretion signal; Sequence can be through from the nucleotide sequence derivation VH that measured and the full length nucleotide sequence of VL; And with the full length amino acid sequence comparison of itself and known antibodies VH and VL and confirm [Sequences of Proteins of Immunological Interest, US Dep.Health and Human Services (1991)].
(3) derive from the amino acid sequence analysis in V district of non-human animal's antibody
Full length amino acid sequence about the V district of the H chain of the antibody that comprises secretory signal sequence and L chain; The length of secretory signal sequence and n terminal amino acid sequence can derive out; Through full length amino acid sequence [the Sequences of proteins of Immunological Interest in the V district of the H chain of they and known antibodies and L chain; US Dep.Health and Human Services (proteinic sequence with immunology importance; The U.S., health and human service progress) (1991)] compare, can find the subgroup that they are affiliated.In addition; Can be through aminoacid sequence [the Sequences of proteins of Immunological Interest in the V district of the H chain of they and known antibodies and L chain; US Dep.Health and Human Services (proteinic sequence with immunology importance; The U.S., healthy and human service progress) (1991)] compare, also can find the aminoacid sequence of each CDR in the V district of H chain and L chain.
(4) structure of the carrier of expressing human chimeric antibody
The structure of the carrier of expressing human chimeric antibody; Like what describe in the clauses and subclauses 3 (1); Through in the carrier that humanized antibody is expressed, the cDNA in V district of H chain and L chain that derives from coding non-human animal's antibody clones the upper reaches of gene in C district of H chain and the L chain of entering coding human antibody.For example; The carrier of expressing human chimeric antibody can be through deriving from coding the human animal each cDNA in V district of H chain and L chain of antibody; DNA is connected structure with synthetic; Synthetic DNA comprises 3 '-terminal nucleotide sequence in V district of H chain and the L chain of the antibody that derives from the non-human animal here, 5 '-terminal nucleotide sequence in the H chain of people's antibody and the C district of L chain, and suitable restriction enzyme enzyme recognition site is all arranged at two ends; With the upper reaches of the gene in the C district of H chain of being cloned into them the coding human antibody that contains in the carrier and L chain, this carrier be such as clauses and subclauses 3 (1) the expression vector of humanized antibody of the suitable expression that makes up of description.
(5) structure of the cDNA in coding human CDR-grafted antibody V district
The cDNA in the V district of coding human CDR-grafted antibody H chain and L chain can be like following acquisition.At first, select to be used to transplant the amino acid of framework (below be called " FR ") in V district of H chain and L chain of people's antibody of CDR in V district of H chain and the L chain of the antibody that derives from the non-human animal.As the aminoacid sequence of people's heavy chain of antibody and L chain V district FR, any amino acid can use, as long as they derive from people's antibody.Example comprises DB; The aminoacid sequence of people's heavy chain of antibody of for example registering in the Protein Data Bank and L chain V district FR; Aminoacid sequence [the Sequences of proteins of Immunological Interest of each subgroup of common people's heavy chain of antibody and L chain V district FR; US Dep.Health and Human Services (the proteinic sequence with immunology importance, the U.S., healthy and human service progress) (1991)] etc.In order to prepare the people CDR-grafted antibody of effective active, the preferred high as far as possible aminoacid sequence of selecting with the V district of the H chain of the purpose antibody that derives from the non-human animal and L chain (at least 60% or more) of amino acid sequence homology.
Then, derive from the cdr amino acid sequence in V district of H chain and L chain of non-human animal's purpose antibody, be transplanted to the FR aminoacid sequence in V district of H chain and L chain of people's antibody of selection, the aminoacid sequence in designer CDR-grafted antibody H chain and L chain V district.The frequency that the codon of finding in the consideration antibody gene nucleotide sequence is selected; Change the aminoacid sequence of design into dna sequence dna [Sequences of proteins of Immunological Interest; US Dep.Health and Human Services (proteinic sequence with immunology importance; The U.S., healthy and human service progress) (1991)], the dna sequence dna of design coding human CDR-grafted antibody H chain and L chain V region amino acid sequence.Dna sequence dna with design is the basis, and the synthetic DNA of synthetic several kinds of about 100 bases of length uses them to carry out PCR.In the case, the reaction efficiency of considering PCR and length that can synthetic DNA, every kind of H chain and 4 to 6 kinds of synthetic DNAs of L chain preferred design.
And through importing the recognition site of suitable restriction enzyme at 5 '-end of two ends being present in synthetic DNA, synthetic DNA can be easy to the clone and get into and in clauses and subclauses 3 (1), make up, and is used for the carrier of humanized antibody expression.Behind the PCR; The product cloning of amplification gets into plasmid for example pBluescript SK (-) (Stratagene production) or similar plasmid; Through the method definite kernel nucleotide sequence in the clauses and subclauses 3 (2), obtain to contain the plasmid of dna sequence dna of aminoacid sequence in V district of H chain and L chain of the people CDR-grafted antibody of coding needs.
(6) modification of people's CDR grafted antibody V region amino acid sequence
Known to through only the CDR among nonhuman animal antibody VH and the VL being migrated among people's antibody VH and the VL when producing people's CDR grafted antibody among the FR simply, its antigen-binding activity be lower than the combination of primary nonhuman animal antibody active [BIO/TECHNOLOGY, 9, 266 (1991)].With regard to its reason, the several amino acid residue of considering Fr rather than CDR directly or indirectly with original nonhuman animal antibody VH and VL in antigen-binding activity relevant, they can change and are the different FR amino-acid residues among people's antibody VH and the VL.In order to address this problem; In people's CDR grafted antibody, in the FR of people's antibody VH and VL aminoacid sequence, the amino-acid residue directly related with conjugated antigen; Or through with CDR in amino-acid residue interact or three-dimensional structure and the indirect relevant amino-acid residue of conjugated antigen through keeping antibody; Identified and be modified to a kind of aminoacid sequence that in original nonhuman animal antibody, to find, to increase the antigen-binding activity [BIO/TECHNOLOGY that has been lowered 9, 266 (1991)].
In the generation of people's CDR grafted antibody, the most important thing is how effectively to differentiate amino-acid residue relevant among the FR with antigen-binding activity, make up the three-dimensional structure of antibody like this, and through the X-radiocrystallography [J.Mol Biol, 112,535 (1977)], microcomputer modelling [Protein Engineering, 7, 1501 (1994)] or similar approach analyze.Although the three-dimensional structure information of antibody has been used to produce the generation of people's CDR grafted antibody, the method that is used to produce the people's CDR grafted antibody that can be used for producing all antibody is not also set up now.Therefore, need carry out multiple trial now, for example produce several kinds of modified antibodies of each antibody, illustrate the relation between each modified antibodies and its antibody binding activity.
Modify like 3 (5) described PCR for basis, the modification of people's antibody VH that has selected and the FR aminoacid sequence of VL can use multiple synthetic DNA to accomplish.With regard to the amplified production that obtains through PCR, measure nucleotide sequence to confirm whether to have carried out the target modification according to the method described in 3 (2).
(7) make up the carrier that is used for the expression of people CDR-grafted antibody
The upper reaches of the H chain of the people's antibody through getting into the cDNA clone in the V district of people CDR-grafted antibody H chain that makes up in coding clauses and subclauses 3 (5) and (6) and L chain in the carriers of describing in the coding clauses and subclauses 3 (1) that are used for the humanized antibody expression and the C district gene of L chain, structure is used for the carrier of people CDR-grafted antibody expression.For example; Through in clauses and subclauses 3 (5) and (6), carrying out PCR when making up the V district of H chain and L chain; Import suitable restriction endonuclease recognition sequence in 5 '-end of used two ends of synthetic dna fragmentation; Like this V district cDNA of people CDR-grafted antibody H chain and L chain can clone get into as clauses and subclauses 3 (1) in the upper reaches of gene in C district of H chain and L chain of the carrier coding human antibody that are used for the humanized antibody expression described; They can be expressed with suitable manner by this way, have therefore made up the expression vector of people's CDR grafted antibody.
(8) the stable humanized antibody that produces
Through importing suitable zooblast to the carrier of the expression humanized antibody of describing in clauses and subclauses 3 (4) or (7), can obtain the stable transformant that produces people's chimeric antibody and people CDR-grafted antibody (both is called " humanized antibody " hereinafter).
The method of the carrier transfered cell of expressing humanized antibody comprise electroporation [No. 257891/90, the patented claim of day examination of the present disclosure, Cytotechnology (electroporation), 3, 133 (1990)] etc.
Zooblast as the carrier of expressing humanized antibody imports can use any cell, so long as can produce the zooblast of humanized antibody.
Example comprises murine myeloma cell, for example NS0 cell and SP2/0 cell, Chinese hamster ovary cell, for example CHO/dhfr-cell and CHO/DG44 cell; Rat myeloma cell, for example YB2/0 cell and IR983F cell; Derive from the bhk cell of Syria hamster kidney; Human myeloma cell is Namalwa cell etc. for example.Preferred Chinese hamster ovary cell CHO/DG44 cell, rat myeloma cell YB2/0 cell and the cell described in 1.
After importing the carrier of expressing humanized antibody; According to disclosed method in the number of patent application 257891/90 of day examination of the present disclosure; Use be used for animal cell culture for example contain G418 vitriol (below be called " G418 "; By SIGMA preparation) substratum, selection can be stablized the transformant that produces humanized antibody.Substratum as animal cell culture; What can mention is RPMI 1640 substratum (Nissui Pharmaceutical productions); GIT substratum (Nihon Pharmaceutical production), EX-CELL 302 substratum (JRH production), IMDM substratum (GIBCO BRL production); Hybridoma-SFM substratum (GIBCO BRL production) adds the for example substratum that obtains of foetal calf serum (below be called " FCS ") etc. of multiple additives in these substratum.Through in substratum, cultivating the transformant that obtains, in substratum, produce and the accumulation humanized antibody.In the substratum generation of humanized antibody and antigen-binding activity can use for example EUSA [below be called " ELISA "; Antibodies:A Laboratory Manual (laboratory manual); Cold Spring Harbor Laboratory; The 14th chapter (1998), Monoclonal Antibodies:Principles and Practice, Academic Press (U.S. academic press) Limited (1996)] etc. method measure.And, according to disclosed method in No. 257891/90, the patented claim of day examination of the present disclosure, use DHFR gene amplification system, the output that produces humanized antibody through transformant is increased.，
Use albumin A post [Antibodies:A Laboratory Manual (antibody: Cold Spring Harbor Laboratory (cold spring harbor laboratory) laboratory manual); The 8th chapter (1988); Monoclonal Antibodies Principles and Practice (monoclonal antibody: principle is in putting into practice); Academic Press (U.S. academic press) Limited (1996)], purifying humanized antibody from the supernatant of culture medium of transformant.In addition, also can use the purification process that generally is used for protein purification.For example, through gel-filtration, ion exchange chromatography, ultrafiltration combines and purifies.Can measure the H chain of the humanized antibody of purifying respectively, the molecular weight of L chain and whole antibody molecule, for example, through polyacrylamide gel electrophoresis [below be called " SDS-PAGE ", Nature (nature), 227, 680 (1970)], Western blotting [Antibodies A Laboratory Manual (antibody: laboratory manual), the 12nd chapter (1988), Monoclonal Antibodies (monoclonal antibody)] etc.
B. prepare the Fc fusion rotein
(1) makes up Fc Expression of Fusion Protein carrier
Fc Expression of Fusion Protein carrier is a kind of animal cell expression carrier that has been inserted into the Fc district of coding human antibody and has wanted Fused proteinic gene, and its structure can pass through the Fc district of coding human antibody and want in the expression vector of Fused proteinic each gene clone precession thing cell.
The Fc district of people's antibody comprises that those except the zone of containing CH2 and CH3 district, contain the part of hinge area and/or the zone of CH1.Also can be any Fc district,, replace, add or insertion to have the activity that combines in fact with Fc γ acceptor as long as at least one amino acid of CH2 or CH3 is deleted.
As the Fc district of coding human antibody with want Fused proteic gene, can use by exon with include molecular chromosomal DNA, also can use cDNA.The method that connects gene and Fc district comprises uses the PCR (molecular cloning) of each gene order as template, Current Protocols in Molecular.Biology (modern molecular biology experimental technique), supplementary issue 1-34).
As the expression vector of zooblast, can use any carrier, as long as the gene of coding human Antibody C region is inserted into wherein and expression therein.Example comprise pAGE107 [Cytotechnology (electroporation), 3, 133 (1990)], pAGE1OS [J.Biochem. (biological chemistry impurity), 101, 1307 (1987)], pHSG274 [Gene (gene), 27, 223 (1984)], pKCR [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 78, 1527 (1981), pSG1 p d2-4 [Cytotechnology (electroporation), 4, 173 (1990)] and analogue.Promotor in the animal cell expression carrier and enhanser contain SV40 early promoter and enhanser [J.Biochem. (biological chemistry impurity), 101,1307 (1987)], moloney murine leukemia virus LTR promotor [Biochem.Biophys.Res.Commun., 149,960 (1987)], IgH chain promotor [Cell (cell), 41, 479 (1985)] and enhanser [Cell (cell), 33, 717 (1983)], and analogue.
(2) prepare coding human antibody Fc district and want Fused proteic DNA
Coding human antibody Fc district with to can be obtained in the following manner by the DNA of fusion rotein.
The synthetic cDNA of mRNA that extracts from the cell or tissue of expressing the target protein that will merge with Fc.Synthetic cDNA is cloned in the carrier like phage or plasmid to obtain the cDNA library.The recombinant phage or the recombinant plasmid that contain the cDNA of the target protein of encoding separate from the library as probe through application target protein part gene order.Measure the full length nucleotide sequence of purpose antibody on recombinant phage or the recombinant plasmid, derive full length amino acid sequence from nucleotide sequence.
As the non-human animal, can use any animal such as mouse, rat, as long as hamster or rabbit are can be from wherein taking out cell or tissue.
The method for preparing total RNA from cell or tissue comprise guanidine thiocyanate-trifluoracetic acid caesium method [Methods in Enzymology, 154,3 (1987)] method that and similar approach, prepares mRNA from total RNA comprises plain post method (molecular cloning, second edition) of oligomerization (dT)-anchoring fiber and similar approach.In addition, the test kit for preparing mRNA from cell or tissue comprises Fast Track mRNA separating kit (Invitrogen manufacturing), Quick Prep mRNA purification kit (Pharmacia manufacturing) and similar approach.
Synthetic cDNA comprises ordinary method (molecular cloning), Current Protocols in Molecular.Biology (modern molecular biology experimental technique), supplementary issue 1-34 with the method for preparing the cDNA library); Use commercial reagent box such as Superscript TM, cDNA synthetic and the pUC pUC (GIBCO BRL manufacturing) of plasmid clone or the method for ZAP-cDNA synthetic agent box (StrataGene (gene) manufacturing); And similar approach.
In the process of preparation cDNA library, the carrier that has been inserted into cDNA can be any carrier, as long as can insert cDNA, this cDNA is to use the mRNA that extracts from cell or tissue as the template synthetic.Example comprise ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research (nucleic acids research), 17, 9494 (1989)], XzapII (StrataGene (gene) manufacturing), λ gt10 and λ gt11 [dna clone, hands-on approach, I, 49 (1985)], Lambda BlueMid (Clontech manufacturing), AExCell (cell), pT7T3 18U (Pharmacia manufacturing), pcD2 [Mol.Cell Biol, 3, 280 (1983)], pUC18 [Gene (gene), 33, 103 (1985)] and analogue.
As being imported into from the intestinal bacteria in the cDNA library of phage or plamid vector construction, can use any intestinal bacteria, as long as can import, express and maintenance cDNA library.Example comprise XL1-Blue MRF ' [Strategies, 5, 81 (1992)], C600 [Genetics (genetics), 39, 440 (1954)], Y1088 and Y1090 [Science (science), 222, 778 (1983)], NM522 [J.Mol.Biol., 166, 1 (1983)], K802 [J.Mol.Biol., 16, 118 (1966)], JM105 [Gene (gene), 38, 275 (1985)] and analogue.
As the cDNA clone's who from the cDNA library, selects the coding target protein method, can use the colony hybridization or the plaque hybridization (molecular cloning, second edition) that adopt isotropic substance or fluorescently-labeled probe.The cDNA of coding target protein also can be according to PCR, and through the preparation primer, use synthetic cDNA from mRNA or cDNA library prepares as template.
The method of target protein and the fusion of people's antibody Fc district is comprised PCR.For example, 5 ' terminal and 3 ' tip designs synthetic oligo DNA (primer) in the gene order of coding target protein carries out PCR to prepare the PCR product.With same method, the gene order design primer in the people's antibody Fc district that will merge for encoding is with preparation PCR product.At this moment, design primer, between the 5 ' end of wanting Fused proteic PCR product 3 ' end and Fc district PCR product, have identical Restriction Enzyme site or identical gene order with following mode.When the amino acid around the needs modification connection site, the primer that is imported into sudden change through use imports sudden change.Further through using two kinds of acquired PCR fragments to carry out PCR to connect gene.They also can be connected through ligation after handling with identical Restriction Enzyme.
Confirming of the nucleotide sequence of DNA can be through the gene order of using suitable Restriction Enzyme digestion to connect with above-mentioned method; Dna fragmentation is cloned in the plasmid like pBluescript SK (-) (StrataGene (gene) manufacturing); Use people's such as normally used nucleotide sequence analysis method such as Sanger dideoxy method [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA 74, 5463 (1977)] or automatically nucleotide sequence analysis appearance such as A.L.F.DNA sequenator (Pharmacia manufacturing) are analyzed.
The full length amino acid sequence of the Fc fusion rotein that contains secretion signal no matter whether acquired cDNA encodes; Sequence can be through the full length nucleotide sequence from the nucleotide sequence derivation Fc fusion rotein measured, and with itself and the comparison of purpose aminoacid sequence and confirm.
(3) the stable Fc fusion rotein that produces
Can stablize the transformant that produces the Fc fusion rotein obtains through importing in the suitable zooblast at the Fc fusion protein expression vector described in (1).
With Fc fusion protein expression vector the method in the zooblast of importing comprise electroporation [Japan has announced No. 257891/90, the patented claim of not substantive examination, Cytotechnology (electroporation), 3, 133 (1990)] and similar approach.
As the zooblast that is imported into the Fc fusion protein expression vector, can use any cell, as long as it is the zooblast that can produce the Fc fusion rotein.
Preferred example comprises murine myeloma cell such as NS0 cell and SP2/0 cell; Chinese hamster ovary cell such as CHO/dhfr-cell and CHO/DG44 cell, rat myeloma cell such as YB2/0 cell and IR983F cell are from the bhk cell of Syria hamster kidney; Human myeloma cell such as Namalwa cell; With similar cell, preferred Chinese hamster ovary cell CHO/DG44 cell, rat bone myeloma YB2/0 cell and the host cell that in the inventive method described in 1, uses.
After importing the Fc fusion protein expression vector; The method described in No. 257891/90, the patented claim of not substantive examination of having announced according to Japan contains the Zooblast culture medium just like G418 and similar reagents through use, and selection can be stablized the selection of the transformant that produces the Fc fusion protein expression vector.The substratum of culture of animal cells comprises RPMI 1640 substratum (Nissui Pharmaceutical manufacturing); GIT substratum (Nihon Pharmaceutical manufacturing); EX-CELL302 substratum (JRH manufacturing); IMDM substratum (GEBCO BRL manufacturing), Hybridoma-SFM substratum (GIBCO BRL manufacturing).Through in these substratum, adding substratum and the similar substratum that several additives such as foetal calf serum obtain.Can in culture supernatant liquid, produce and accumulate the Fc fused protein through in substratum, cultivating acquired transformant.The generation of Fc fusion rotein and antigen-binding activity can be measured through the method like ELISA in culture supernatant liquid.Also can adopt dhfr gene amplification system to increase the amount of the Fc fusion rotein that transformant produces according to the method for describing in No. 257891/90, the patented claim of having announced not substantive examination in Japan.
Can adopt albumin A post purifying Fc fusion rotein (antibody, the 8th chapter from the culture supernatant liquid of transformant; Monoclonal antibody).In addition, also can use the purification process that is used as protein purification usually.For example, can be through uniting the use gel-filtration, purifying is carried out in ion-exchange chromatography and ultrafiltration.As the Fc fusion protein molecule of whole purifying, molecular weight through SDS-PAGE [Nature (nature), 227,680 (1970)], Western blotting [antibody, the 12nd chapter, (1988), monoclonal antibody] or similar approach are measured.
Therefore, adopt zooblast to be described as the method that host cell produces antibody compositions, but as stated; Antibody compositions also can pass through yeast; Insect cell, vegetable cell, animal individual or plant individual produce in zooblast through identical method.
When cell innately has the ability of expressed antibody molecule, the method that the generation of purpose antibody compositions can be employed in described in 1 produces the cell of antibody, culturing cell, antibody purification compsn from the substratum that obtains then through preparation.
4. the activity rating of antibody compositions
As the measuring method of the effector functions of amount, antibody purification and the antigen bonded activity of antibody purification compsn and antibody purification compsn, can use the currently known methods of describing in the documents such as monoclonal antibody, antibody engineering.
For example, when antibody compositions is humanized antibody, its with antigenic combine active and with the cultivation clone of antigen positive combine activity can through as the method detection of ELISA and immunofluorescence technique [Cancer Immunol.Immunother., 36, 373 (1993)].To antigen positive cultivate cytotoxic activity (after this being called " CDC activity ") that clone's cytotoxic activity can be through measuring complement-dependences, ADCC activity [Cancer Immunol.Immunother., 36, 373 (1993)] wait and estimate.
In addition, antibody compositions can be estimated through use suitable animal species model such as the Macaca fascicularis that is bordering on the people that connect in the security of philtrum and curative effect.
5. the analysis of sugar chain in the antibody compositions
The sugar chain structure of the antibody molecule of in various cells, expressing can be analyzed according to the general analysis of the sugar chain structure of gp.For example, the sugar chain that is incorporated into the IgG molecule comprises for example semi-lactosi of neutral sugar chain, seminose or Fucose; Aminosugar is the N-acetylglucosamine for example; Acid sugar is sialyl for example, can be through for example using methods analysts such as sugar compsn analyzing sugar chain structure, two-dimentional sugar chain collection of illustrative plates.
(1) analysis of neutral sugar and aminosugar composition
The sugar chain of antibody compositions can through sugar chain with for example trifluoroacetic acid acidolysis of acid, discharge neutral sugar and aminosugar, measure composition ratios and analyze.
Example comprises the method for use by the sugar compsn analyser (BioLC) of Dionex production.BioLC be a kind of with HPAEC-PAD (high performance anion exchange chromatography method pulsed current mensuration) [J Liq.Chromatogr. (liquid chromatography magazine), 6, 1577 (1983)] and analyze the instrument of sugar compsn.
The ratio of compsn also can be used the fluorescent marker method analysis of using the 2-EL-970.Concrete, the fluorescent mark of the sample of acidolysis with the 2-EL-970ization, HPLC analysed compsn then, the ratio of compsn can according to currently known methods [Agric.Biol Chem. (agricultural biochemistry), 55(1), 283-284 (1991)] calculate,
(2) analysis of sugar chain structure
The sugar chain structure of antibody molecule can use the analysis of two-dimentional sugar chain figure spectrometry [Anal Biochem. (agricultural biochemistry), 17173 (1988), Biochemical Experimentation Methods 23-Methods for.Studying Glycoprotein Sugar Chains (method of Biochemistry Experiment method 23-research glycoprotein candy chain) (Japan Scientific Societies Press (Japanese science association publishes)) edits (1989) by Reiko Takahashi].Two dimension sugar chain figure spectrometry be through; For example; The residence time of the sugar chain that obtains with reverse-phase chromatography respectively or wash-out position are as the X axle; Compare them as the y axle in the residence time of the sugar chain that normal phase chromatography obtains or wash-out position with the result of known sugar chain, thus the method for derivation sugar chain structure.
Concrete, the antibody hydrazinolysis, sugar chain discharges from antibody, the sugar chain of release with 2-EL-970 (below be called " PA ") fluorescent mark [J.Biocbcni., 95, 197 (1984)], sugar chain is through gel-filtration then, and reverse-phase chromatography, from excessive PA-reagent treatment, separates.Then, each peak of isolating sugar chain separates through normal phase chromatography.Through marking and drawing the result that obtains on two-dimentional sugar chain collection of illustrative plates, they with sugar chain standard point (by Takara Slluzo drafting) or document [Anal Biochem., 171, 73 (1988)] relatively derive sugar chain structure.
Structure through two-dimentional sugar chain atlas calculation is derived can further be used mass spectroscopy, for example the MALDI-TOF-MS of each sugar chain conclusive evidence.
6. the application of the antibody compositions that obtains among the present invention
It is active that the antibody compositions that obtains among the present invention has high ADCC.Have the active antibody of high ADCC and can be used for preventing and treating various diseases, comprise cancer, diseases associated with inflammation, Immunological diseases such as autoimmune disorders, transformation reactions etc., cardiovascular disorder and various by the disease that causes like virus and infection.
In cancer is under the malignant tumour situation, and cancer cell increases.The growth of general antitumour drug anticancer.On the contrary, having the active antibody of high ADCC can treat cancer through its cell killing effect infringement cancer cells, is than the more effective therapeutical agent of general antitumour drug therefore.At present, in novel remedies for cancer, the independent antitumous effect of antibody drug is not enough, therefore carried out with the combination therapy of chemotherapy [Science (science), 280, 1197 (1998)].If finding has higher antitumous effect with antibody compositions of the present invention separately, the dependency to chemotherapy will descend so, and spinoff will reduce.
In Immunological diseases such as diseases associated with inflammation, autoimmune disorders and transformation reactions; The body internal reaction of disease is to be caused by the medium molecule that immunocyte discharges, and therefore can have the active cleaning antibody immunocyte of high ADCC through employing suppresses transformation reactions.
Cardiovascular disorder comprises arteriosclerosis etc.At present, arteriosclerosis adopts the balloon catheter treatment, has the active antibody of high ADCC and can grow and prevent and treat cardiovascular disorder through suppressing arterial cell in the restenosis of balloon catheter treatment back but adopt.
Having the active antibody of high ADCC through employing suppresses can be prevented and treat the various diseases that comprises virus and infectation of bacteria by the propagation of the cell of virus or infectation of bacteria.
The antibody of the antibody of the antibody of identification taa, identification transformation reactions or inflammation related antigen, identification cardiovascular disorder related antigen and the antibody of identification virus or infectation of bacteria related antigen illustrate below.
The antibody of identification taa comprises: anti-GD2 antibody [Anticancer Res. (anticancer research), 13, 331-336 (1993)], anti-GD3 antibody [Cancer Immunol.Immunother., 36, 260-266 (1993)], anti-GM2 antibody [Cancer Res. (cancer research), 54, 1511-1516 (1994)], Anti-HER 2 [Proc.Natl.Acad Sci.USA, 89, 4285-4289 (1992)], anti-CD 52 antibody [Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 89, 4285-4289 (1992)], anti-MAGE antibody [British J.Cancer (cancer), 83, 493-497 (2000)], anti-HM1.24 antibody [Molecular Immunol. (molecular immunology), 36, 387-395 (1999)], anti-parathyroid hormone-related protein (PTHrP) antibody [Cancer (cancer), 88, 2909-2911 (2000)], anti-FGF8 antibody (Proc.Natl.Acad.Sci. (Proc. Natl. Acad. Sci.USA) USA, 86, 9911-9915 (1989), alkali-resistivity fibroblast growth factor antibody and anti-FGF8 receptor antibody (J.Biol.Chem, 265, 16455-16463 (1990)), alkali-resistivity fibroblast growth factor acceptor antibody and synalbumin like growth factor antibody [J.Neurosci.Res., 40, 647-659 (1995)], anti-IGFR body antibody [J.Neurosci.Res., 40, 647-659 (1995)], anti-PMSA antibody [J.Urology (urology research), 160, 2396-2401 (1998)], anti-vascular endothelial cell growth factor antibody [Cancer Res. (cancer research), 57, 4593-4599 (1997)], anti-vascular endothelial cell growth factor receptor 2 body antibody [OncoGene, 192138-2146 (2000)], anti-CA 125 antibody, anti-17-1A antibody, anti-plain av β 3 antibody, anti-CD 33 antibody, anti-CD22 antibody, anti-hla antibody, anti-HLA-DR antibody, anti-CD20 antibodies, anti-CD 19 antibodies, anti-EGF receptor antibody (the Immunology Today (immunology today) of integrating 21(8), 403-410 (2000)), anti-CD10 antibody [American Journal of Clinical Pathology (U.S.'s clinical pathology magazine), 113, 374-382 (2000)], etc.
The antibody of identification transformation reactions or inflammation related antigen comprises: anti-interleukin 6 antibody [Immunol.Rev. (immunology research), 127, 5-24 (1992)], anti-interleukin 6 receptor antibody [Molecular Immunol. (molecular immunology), 31, 371-381 (1994)], anti-interleukin-15 antibody [Immunol.Rev (immunology research)., 127, 5-24 (1992)], anti-interleukin-15 receptor antibody and anti-interleukin 4 antibody [Cytokine (cytokine), 3, 562-567 (1991)], anti-interleukin 4 receptor antibodies [J.Immunol. (Journal of Immunology) Meth., 217, 41-50 (1998)], D2E7 [Hybridoma, 13, 183-190 (1994)], the anti-tumor necrosis factor receptor antibody [Molecular Pharmacol, 58, 237-245 (2000)], anti-CCR4 antibody [Nature (nature), 400, 776-780 (1999)], anti-cytokine antibodies [J.Immuno.Meth, 174,249-257 (1994)], antibacterial agent receptor antibody [J.Exp.Med. (experimental technique magazine), 186, 1373-1381 (1997)], anti-IgE antibodies, anti-CD23 antibody, anti-CD11a antibody [Immunology Today (immunology today), 21(8)., 403-410 (2000)], anti-CRTH2 antibody (J Immunol., 162, 1278-1286 (1999)), anti-CCR8 antibody (WO99/25734), anti-CCR3 antibody (US6207155), etc.
The antibody of identification cardiovascular diseases related antigen comprises: anti-GpIIb/IIIa antibody [J.Immunol., 152.2968-2976 (1994)], antiplatelet deutero-growth factor antibodies [Science (science), 253, 1129-1132 (1991)], antiplatelet deutero-growth factor receptor antibody [J.Biol.Chem. (journal of biological chemistry), 272, 17400-17404 (1997)] and anticoagulin antibody [Circulation (circulation), 101, 1158-1164 (2000)], etc.
The antibody of identification autoimmune disorders related antigen comprises: anti-from body-dna antibody [Immunol.Letters, 72,61-68 (2000)], etc.
The antibody of identification virus or infectation of bacteria related antigen comprises: anti-gp120 antibody [Structure (structure), 8, 385-395 (2000)], anti-CD 4 antibodies [J.Rheumatology (rheumatology magazine), 25, 2065-2076 (1998)], anti-CCR4 antibody, anti-Vero toxin antibody [J.Clin.Microbiol, 37396-399 (1999)], anti-autoimmune disorders (psoriasis, rheumatic arthritis, Crohn disease, ulcerative colitis, systemic lupus erythematous, multiple sclerosis; Deng) antibody, anti-CDlla antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-cd 3 antibodies, anti-CD 4 antibodies, anti-integrin alpha 4 β 7 antibody, anti-GD40L antibody, anti-IL-2 antibody [Immunology Today (immunology today) 21(8), 403-410 (2000)], etc.
These antibody can obtain from community organization; RIKEN gene pool like ATCC (The American Type Culture Collection), The Institute of Physical and Chemical Research; With National Institute of BioScience and Human Technology; Agency of Industrial Science and Technology, or be obtained from all reagent sales companys such as Dainippon Pharmaceutical, R&D SYSTEMS; PharMingen, Cosmo Bio and Funakoshi.
Antibody compositions of the present invention can be used as independent treatment reagent administration.Usually, preferably antibody compositions mixes with at least a pharmaceutically acceptable carrier, and as pharmaceutical formulation, known appropriate method prepares in the technical field with the preparation pharmacy it.
Preferably select the most effective route of administration in the treatment.Example comprises oral, parenterai administration, for example the oral cavity, tracheae, rectum, subcutaneous, muscle, intravenous using.In Antibody Preparation, intravenous administration is preferred.
Agent shape comprises spraying, capsule, tablet, particle, syrup, emulsion, suppository, injection, ointment, sticking paste etc.
The example that is appropriate to oral pharmaceutical formulations comprises emulsion, syrup, capsule, tablet, powder, particle or the like.
Liquid preparation, example emulsion and syrup can be used like additive, water; Sugar is sucrose, sorbyl alcohol and fructose for example; Divalent alcohol is polyoxyethylene glycol and Ucar 35 for example; Oil is til, sweet oil and soya-bean oil for example; Sanitas is the p-p-Hydroxybenzoate for example; For example preparation such as strawberry flavor and peppermint of spices.
Glue, tablet, powder, particle etc. can be used additive, and vehicle is lactose, glucose, sucrose and N.F,USP MANNITOL for example; Decomposition agent is starch and Sodium L-arginate for example; Lubricant is Magnesium Stearate and talcum for example; Tackiness agent is Z 150PH, TSK-Gel G 2000HXL and gelatin for example, and tensio-active agent is fatty ester for example, for example USP Kosher preparation of plasticizer.
The pharmaceutical formulations that is fit to parenterai administration comprises injection, suppository, sprays or the like.
Injection can use carrier for example salts solution, glucose solution or their mixture or the like.Injectable powder can be with the cryodesiccated antibody compositions of ordinary method preparation, toward wherein adding the sodium-chlor preparation.
Suppository can use carrier, for example theobroma oil, hydrogenated fat or carboxylic acid preparation.
Spraying also can be used such antibody compositions or use does not stimulate patient oral cavity or tunica mucosa tracheae, and the preparing carriers that the enhancing antibody compsn absorbs through being dispersed into fine particle to it.
Carrier comprises lactose, glycerine etc.According to the character of antibody compositions and carrier, can prepare pharmaceutical formulations for example sprays and dry powder.In addition, the composition as the examples of additives of oral prepns also can add in the parenteral preparation.
Though clinical dosage or frequency of administration according to therapeutic interest effect, application process, treatment cycle, age, body weight or the like difference, generally are that 10 microgram/kilograms are to 20 micrograms/kg/day/people.
And as the method for check antibody compositions to the antitumor action of different tumour cells, external check comprises CDC activity test method, ADCC activity test method etc.; Check is included in the anti-tumor experiment etc. that laboratory animal is for example used the tumour system in the mouse in the body.
Active and ADCC determination of activity of CDC and anti-tumor experiment can be according to cancer Immunology Immunotherapy, 36, 373 (1993); Cancer Research, the method for describing in 54,1511 (1944) etc. is carried out.
The present invention will describe in detail as follows according to experimental example; Yet experiment only is simple declaration of the present invention, and scope of the present invention is not limited.
Brief Description Of Drawings
Fig. 1 shows that the anti-CCR4 chimeric antibody that is produced by the cell-derived clone of rat bone myeloma YB2/0 KM2760#58-35-16 and clone 1-15 is active to the ADCC of CCR4/EL-4 cell.Ordinate zou and X-coordinate be showed cell cytotoxic activity and AC respectively, the activity of the anti-CCR4 chimeric antibody KM2760-1 that " n " and " A " represent respectively to be produced by clone KM2760#58-35-16 active and the anti-CCR4 chimeric antibody KM2760-2 that produced by clone 1-15.
Fig. 2 shows that the PA that from the antibody that the clone who imports mfFUTS-6 and pAGE249 produces, prepares handles the type of elution of sugar chain, obtains through its reversed-phase HPLC analysis.Fig. 2 A and Fig. 2 B show that respectively the type of elution of the PA processing sugar chain that from the antibody that the clone who imports mfFUT8-6 produces, prepares and the PA that from the antibody that the clone who imports pAGE249 produces, prepares handle the type of elution of sugar chain.Ordinate zou and X-coordinate show relative intensity of fluorescence and elution time respectively.
Fig. 3 shows the FUT8 of employing RT-PCR mensuration in each host clone and the photo of beta-actin transcription product level.Use the cDNAs that can produce the clone KM2760#58-35-16 of KM2760-1, the clone 1-15 that can produce KM2760-2 and parental generation rat bone myeloma YB2/0 cell preparation as template; The primer special with FUT8 carries out PCR to (SEQ ID NO:13 and 14) or the special primer of beta-actin to (SEQ ID NOs:11 and 12), and through reaction soln is carried out the result that agarose gel electrophoresis provides gained.
Fig. 4 shows the structure of plasmid ploxPPuro.
Fig. 5 shows the structure of plasmid pKOFUT8gE2-1.
Fig. 6 shows the structure of plasmid pKOFUT8gE2-2.
Fig. 7 shows the structure of plasmid pscFUT8gE2-3.
Fig. 8 shows the structure of plasmid pKOFUT8gE2-3.
Fig. 9 shows the structure of plasmid pKOFUT8gE2-4.
Figure 10 shows the structure of plasmid pKOFUT8gE2-5.
Figure 11 shows the structure of plasmid pKOFUT8Puro.
Figure 12 shows that as α 1 the CHO clone's of 6-fucosyl transferase gene fracture clone Ist Δ FUT8 2-46-1 and the genome Southern of clone 2-46-H10 analyze photo.
Figure 13 shows that as α 1 the CHO clone's of 6-fucosyl transferase gene fracture clone Ist Δ FUT8 2-46 and the genome Southern of clone Ist Δ FUT8 2-46-H10 analyze photo.
Figure 14 shows that purifying is active from the ADCC of FUT8 allelotrope fracture clone's anti-CCR4 chimeric antibody.Ordinate zou and X-coordinate be showed cell cytotoxic activity and AC respectively." ▲ " and " ■ " represent respectively from the active of the Chinese hamster ovary celI clone 5-03 deutero-antibody purification that produces anti-CCR4 chimeric antibody with from cloning the activity of Ist.FUT8 2-46-1 deutero-antibody purification.
Figure 15 shows the structure of plasmid Pkofut8Neo.
Figure 16 shows that clone gene group Southern analyzes photo, and one of allelic copy of CHO/DG44 cell FUT8 ruptures among the clone.
Figure 17 shows that clone gene group Southern analyzes photo, and two of the CHO/DG44 cell PUTS allelotrope all rupture among the clone.
Figure 18 shows that clone gene group Southern analyzes photo, and drug resistant gene is removed from two PUTS allelotrope of CHO/DG44 cell among the clone.
Figure 19 shows the structure of plasmid pBS-2B8L.
Figure 20 shows the structure of plasmid pBS-2B8H and plasmid pBS-28B8Hm.
Figure 21 shows the structure of plasmid pKANTEX2B8P.
Figure 22 shows that the CD 20 antagonizing Chimeric antibody of purifying from the CHP/DG44 clone that the FUT8 Gene Double knocks out is the ADCC activity of Raji cell to the human B lymphocyte culturing cell.Ordinate zou and X-coordinate be showed cell cytotoxic activity and AC respectively.
Figure 23 shows the structure of plasmid CHfFUT8-pCR2.1.
Embodiment of the present invention
Embodiment 1
Produce the cell of the anti-CCR4 chimeric antibody of stably manufactured:
Adopt the tandem expression vector pKANTEX2160 of the anti-CCR4 chimeric antibody of describing among the WO 01/64754 to prepare as follows, cell that can the anti-CCR4 chimeric antibody of stably manufactured.
(1) produces the cell of antibody with rat bone myeloma YB2/0 cell preparation
Through electroporation the anti-CCR4 chimeric antibody expression vector of 10 μ g pKANTEX2160 is imported 4 * 10 6Rat bone myeloma YB2/0 cell (ATCC CRL 1662) [Cytotechnology (electroporation), 3133 (1990)] after; Cell suspension in 40mlHybridoma-SFM-FBS (5) [Hybridoma-SFM substratum (being produced by Invitrogen) has added 5%FBS (being produced by PAA Laboratories)], and is distributed into 96 well culture plates (being produced by Sumitomo Bakelite) with 200 μ l/ holes.At 5%CO 2Cultivate after 24 hours for 37 ℃ in the incubator, add G418 to 1mg/ml concentration, cultivated for 1 to 2 week subsequently.Observe the transformant growth that shows the G418 tolerance through colony formation, from this hole, reclaim the culture supernatant thing, the ELISA that describes in (2) item through embodiment 2 measures the antigen-binding activity of anti-CCR4 chimeric antibody in the supernatant thing.
For the transformant of observing in the culture supernatant thing in the hole that produces anti-CCR4 chimeric antibody; In order to increase the amount of producing antibody with DHFR gene amplification system, each is suspended in Hybridoma-SFM-FBS (5) substratum that has added 1mg/ml G418 and 50nM DHFR suppressor factor MTX (being produced by SIGMA) to obtain 1-2 * 10 with them 5The density of cell/ml, this suspension-s distributes in the hole of into 24 holes dull and stereotyped (by Greiner production) with 1ml.At 5%CO 2In the incubator 37 ℃ cultivated for 1 to 2 week after, induced the transformant that shows 50nM MTX tolerance.Observing in the hole of transformant growth the ELISA that describes in (2) through embodiment 2 of the antigen-binding activity of anti-CCR4 chimeric antibody in the culture supernatant thing measures.
For the transformant of observing in the culture supernatant thing in the hole that produces anti-CCR4 chimeric antibody; Improve MTX concentration through identical method; Final acquisition can be grown having added on Hybridoma-SFM-FBS (5) substratum of 200nM MTX, and can highly produce the transformant of anti-CCR4 chimeric antibody.The transformant that obtains is processed unicellular (clone) twice through the restriction dilution, and the clone that will obtain is called clone KM2760#58-35-16.In the case, adopt the α 1 that describes among the WO00/61739, the measuring method of 6-fucosyltransferase (after this being called " FUT8 ") gene transcript is selected to produce the clone of transcription product relatively in a small amount, and is used as suitable clone.
(2) produce the cell of antibody with the CHO/DG44 cell preparation
Through electroporation the anti-CCR4 chimeric antibody expression vector of 4 μ g pKANTEX2160 is imported 1.6 * 10 6The CHO/DG44 cell [Cytotechnology (electroporation), 3133 (1990)] after; Cell suspension in 10ml IMDM-dFBS (10)-HT (1) [IMDM substratum (being produced by Invitrogen) has added the HT additive (being produced by Invitrogen) of 10%dFBS (being produced by Invitrogen) and 1 * concentration], and is distributed into 96 well culture plates (by Iwaki Glass production) with 100 μ l/ holes.At 5%CO 2Cultivate after 24 hours for 37 ℃ in the incubator, substratum is changed into IMDM-dFBS (10) (adding the IMDM substratum of 10% dialysis FBS), cultivates for 1 to 2 week subsequently.Reclaim the culture supernatant thing from observing to have formed the hole that shows the transformant growth that does not rely on HT, the ELISA that describes in (2) item through embodiment 2 measures the amount that anti-CCR4 chimeric antibody produces in the supernatant thing.
For the transformant of observing in the culture supernatant thing in the hole that produces anti-CCR4 chimeric antibody; In order to increase the amount of producing antibody with DHFR gene amplification system, each is suspended in IMDM-dFBS (10) substratum that has added 50nM MTX to obtain 1-2 * 10 with them 5The density of cell/ml, this suspension-s distributes in the hole of into 24 holes dull and stereotyped (by Iwaki Glass production) with 0.5ml.At 5%CO 2In the incubator 37 ℃ cultivated for 1 to 2 week after, induced the transformant that shows 50nM MTX tolerance.For the transformant in the hole of observing growth, MTX concentration is brought up to 200nM through identical method, and final acquisition can be grown having added on IMDM-dFBS (10) substratum of 200nM MTX, and transformant that can the anti-CCR4 chimeric antibody of mass production.The transformant that obtains is called clone 5-03.
Embodiment 2
The comparison of the antibody compositions that the antibody compositions that the cell of the generation antibody that FUT8 genetic expression reduces produces and its parental cell produce:
ADCC between the antibody compositions that the antibody compositions that the adorned cell of icp gene group produces and its parental cell produce is active, and said modification has reduced α 1, the activity of 6-Fucose modifying enzyme.
(1) preparation antibody compositions
Antibody compositions as the adorned antibody producing cell generation of genome; Said modification has reduced α 1; The activity of 6-Fucose modifying enzyme; Use is antibody purified compsn KM2760-1 from KM2760#58-35-16 culture supernatant thing, and wherein the FUT8 gene transcript is described in embodiment 1 (1) item.
The antibody compositions that is produced by parental generation rat myeloma cell YB2/0 cell (ATCC CRL1662) prepares as follows.
Through electroporation the anti-CCR4 chimeric antibody expression vector of 10 μ g pKANTEX2160 (being described among the WO 01/64754) is imported 4 * 10 6Rat bone myeloma YB2/0 cell (ATCC CRL1662) [Cytotechnology (electroporation), 3133 (1990)] after; Cell suspension in 40mlHybridoma-SFM-FBS (5) [Hybridoma-SFM substratum (being produced by Invitrogen) has added 5%FBS (being produced by PAA Laboratories)], and is distributed into 96 well culture plates (being produced by Sumitomo Bakelite) with 200 μ l/ holes.At 5%CO 2Cultivate after 24 hours for 37 ℃ in the incubator, add G418 to concentration be 1mg/ml, cultivated for 1 to 2 week subsequently.Observe the transformant growth that shows the G418 tolerance through colony formation, from this hole, reclaim the culture supernatant thing, measure the antigen-binding activity of anti-CCR4 chimeric antibody in the supernatant thing through the ELISA that describes in present embodiment (2) item.
For the transformant of observing in the culture supernatant thing in the hole that produces anti-CCR4 chimeric antibody; In order to increase the amount of producing antibody with DHFR gene amplification system, each is suspended in Hybridoma-SFM-FBS (5) substratum that has added 1mg/ml G418 and 50nM DHFR suppressor factor MTX (being produced by SIGMA) to obtain 1-2 * 10 with them 5Cell/ml density, this suspension-s distributes in the hole of into 24 holes dull and stereotyped (by Greiner production) with 1ml.At 5%CO 2In the incubator 37 ℃ cultivated for 1 to 2 week after, induced transformant with 50nM MTX tolerance.Observing in the hole of transformant growth the antigen-binding activity of anti-CCR4 chimeric antibody in the culture supernatant thing measures through the ELISA that describes in the present embodiment (2).
For the transformant of observing in the culture supernatant thing in the hole that produces anti-CCR4 chimeric antibody; Improve MTX concentration through identical method; Final acquisition can be grown having added on Hybridoma-SFM-FBS (5) substratum of 200nM MTX, and transformant that can the anti-CCR4 chimeric antibody of mass production.The clone who obtains is called clone 1-15.The dilution of clone 1-15 not-go end system is transformed into unicellular (clone).
The following purifying of anti-CCR4 chimeric antibody by transformant clone 1-15 generation.
The transformant clone 1-15 that expresses anti-CCR4 chimeric antibody is suspended in Hybridoma-SFM (being produced by the Invitrogen) substratum that has added 200nMMTX and 5%Daigo ' s GF21 (being produced by Wako Pure Chemical Industries) to obtain 2 * 10 5Cell/ml density, and adopt revolving bottle (Iwaki Glass production) in 37 ℃ thermostatical storehouse, to carry out the fed-batch jolting and cultivate.Cultivate after 8 to 10 days, with Prosep-A (producing) post and gel-filtration anti-CCR4 chimeric antibody of purifying from the culture supernatant thing that reclaims by Millipore.The anti-CCR4 chimeric antibody of purifying is called clone KM2760-2.
(2) antibody binding activity of antibody compositions
The antibody binding activity of two kinds of antibody compositions that obtain in this embodiment (1) detects through the ELISA of following demonstration.
Select compound 1 (SEQ ID NO:6) as can with zone, the people CCR4 extracellular peptide of anti-CCR4 chimeric antibody reaction.In order in the active detection of ELISA, to use it, gripping thing altogether and being used as antigen through following method preparation and BSA (bovine serum albumin) (producing) by Nacalai Tesque.Promptly; The DMSO solution 100ml that contains 25mg/ml SMCC [4-(N-maleimide methyl)-hexanaphthene-1-formic acid N-hydroxyl succinimide ester] (being produced by Sigma) dropwise adds 900ml and contains in the PBS solution of 10mg BSA under with the wortex device condition of stirring, stirred gently subsequently 30 minutes.As on, add the 1ml reaction soln at gel-filtration column, use 1.5ml PBS wash-out then with 25ml PBS equilibrated NAP-10 post, with the elutant that obtains as BSA-SMCC solution (through the light absorption ratio calculating BSA concentration at 280nM place).Then, 250ml PBS is added in the 0.5mg compound 1, through adding 250ml DMF it is dissolved fully then, under vorticity, add BSA-SMCC solution, stirred gently subsequently 3 hours.Reaction soln is used the PBS dialysed overnight down at 4 ℃, is 0.05% to wherein adding sodium azide to final concentration, and mixture filters through the 0.22mm filter, to be used as BSA-compound 1 solution.
The thing of gripping altogether of preparation distributes into 96 hole EIA plates (being produced by Greiner) with 0.05 μ g/ml and 50 μ l/ holes, and 4 ℃ of night incubation to attach.Behind each hole of PBS flushing, to wherein adding 1%BSA-PBS, make it at room temperature react to block remaining reactive group with 100 μ l/ holes.After washing each hole with the PBS (after this being called " Tween-PBS ") that contains 0.05%Tween20, add the culture supernatant thing of transformant with 50 μ l/ holes, and room temperature reaction 1 hour.After the reaction, with each hole of Tween-PBS flushing, then, goat anti human IgG (γ) antibody-solutions (American Qualex productions) that adds with the peroxidase labelling of 6000 times of 1%BSA-PBS dilutions with 50 μ l/ holes resists as two, and room temperature reaction 1 hour.Reaction also subsequently with after the Tween-PBS flushing, is added the colour developing of ABTS substrate solution with 50 μ l/ holes, and after this 20 minutes, through add 5%SDS solution termination reaction with 50 μ l/ holes.Measure the light absorption ratio at 415nm place thereafter.
The result that above-mentioned ELISA measures is, the KM2760-1 of purifying and KM2760-2 with the CCR4 partial peptide to combine on the activity be similar.
(3) ADCC of the KM2760-1 of purifying and 2760-2 is active.
The KM2760-1 of purifying and the ADCC of 2760-2 are active to be detected as follows.
Anti-CCR4 chimeric antibody is active to the ADCC of the CCR4/EL-4 cell (WO01/64754) of height indicator intelligent CCR4.
(a) preparation target cell suspension liquid
Preparation 1.5 * 10 6Behind the CCR4/EL-4 cell of the expressing human CCR4 that WO 01/64754 describes, be equivalent to the normal radioactivity material of 5.55MBq Na 2 51CrO 4Add wherein, subsequently 37 ℃ of reactions 1.5 hours thereby use the labelled with radioisotope cell.After the reaction, centrifugal again through being suspended in the substratum, cells washed 3 times is suspended in the substratum then on ice 4 ℃ again and hatches 30 minutes with spontaneous decomposition radioactivity material.After centrifugal, regulate cell to 2 * 10 through adding the 15ml substratum 5Cell/ml density, and as target cell suspension liquid.
(b) preparation people effector cell suspension-s
Gather the 60ml peripheral blood from healthy donors, wherein add 0.6ml heparin sodium (producing), mix gently subsequently by Shimizu Pharmaceutical.Adopt Lymphoprep (producing) by AXIS SHIELD according to producer's working instructions centrifugal mixture (800g, 20 minutes) with the separating monocytic cell layer.Through with 3 cells washed of substratum centrifugal (1,400rpm, 5 minutes), be resuspended in then in the substratum to 5 * 10 6Cell/ml density also is used as people effector cell's suspension-s.
(c) detect the ADCC activity
Distribution 50 μ l (1 * 10 in each hole of 96 hole U-shape base plates (producing) by Falcon 4Cells/well) the target cell suspension liquid of preparation in (1) item.Then, add people effector cell's suspension-s 100 μ l (5 * 10 of preparation in (2) item 5Cells/well, effector cell and target cell ratio become 50: 1).In addition, adding every kind of anti-CCR4 chimeric antibody to final concentration is 0.0001 to 10 μ g/ml, and 37 ℃ were reacted 4 hours subsequently.After the reaction, centrifugal flat board also detects in the supernatant thing with γ-telltale 51The amount of Cr.Through carrying out identical step, the single culture base of employing nobody's effector cell's suspension-s and antibody-solutions is also measured in the supernatant thing 51The amount of Cr is calculated spontaneous dissociated 51The Cr amount.Through carrying out identical step, adopt the 1mol/L hydrochloric acid soln but not antibody-solutions and people effector cell's suspension-s and measure in the supernatant thing 51The amount of Cr is calculated always dissociated 51The Cr amount.Calculate ADCC active (%) according to equation (I).
Figure GSB00000760248000661
According to the result that above method is measured, KM2760-1 and KM2760-2's is active significantly different, and the activity of KM2760-2 significantly is lower than KM2760-1 (Fig. 1).
(4) the sugar chain analysis of antibody compositions
The sugar chain analysis of KM2760-1 and KM2760-2 is following.
KM2760-1 and KM2760-2 all accept hydrazinolysis with cracking sugar chain from albumen [Method of Enzymology, 83, 263 (1982)].After removing hydrazine, add ammonium acetate aqueous solution and acetic anhydride and carry out the N-acetylizing through the Evaporation under the reduced pressure.After the freeze-drying, carry out 2-aminopyrimidine fluorescent mark [J.Biochem. (biological chemistry impurity), 95, 197 (1984)].From unnecessary reagent, separate fluorescently-labeled sugar chain group (the sugar chain group that PA-handles) with Superdex Peptide HR 10/30 post (producing) by Pharmacia.With the dry sugar chain cut of centrifugal thickener and be used as the sugar chain group that the PA-of purifying handles.Then, the sugar chain group of the PA-of purifying processing is accepted reversed-phase HPLC analysis (Fig. 2) with CLC-ODS post (Shimadzu production).
As buffer A, sodium phosphate buffer (pH3.8)+0.5%1-butanols is analyzed by following gradient as buffer B with sodium phosphate buffer (pH 3.8).
The peak that shows among Fig. 2 (1) to (8) has following structure.
Figure GSB00000760248000681
GlcNAc, Gal, Man, Fuc and PA represent N-acetylglucosamine, semi-lactosi, seminose, Fucose and pyridinylamino respectively.
Among Fig. 2, no α 1, the area calculating that (1) to (4) accounts for peak (1) to (8) from the peak of the sugar chain group ratio of 6-Fucose, in conjunction with α 1, the sugar chain group ratio of 6-Fucose is from calculating the area calculating that peak (5) to (8) accounts for peak (1) to (8).
KM2760-1 does not have α 1, and the ratio of the sugar chain of 6-Fucose is different from KM2760-2.
(5) antibody producing cell α 1,6-fucosyltransferase expression of gene level
Fig. 3 demonstration is measured according to the method for WO 00/61739 description of embodiment 8; Parental generation rat bone myeloma YB2/0 cell, can produce the clone KM2760#58-35-16 of KM2760-1 and can produce the α 1 of the clone 1-15 of KM2760-2,6-fucosyltransferase and beta-actin gene transcript are measured the result of level.Clone 1-15 produces α 1, and the amount of 6-fucosyltransferase gene is similar with parental generation rat bone myeloma YB2/0 cell, but the amount that clone KM2760#58-35-16 produces obviously is less than other clone.
Confirm that based on The above results the adorned antibody producing cell of genome can produce than its parental cell has the active antibody compositions of higher ADCC, said modification makes its α 1, and 6-Fucose modification enzyme activity is lower or deletion than its parental cell.
Embodiment 3
The Chinese hamster ovary celI of preparation FUT8 gene elmination also uses this cell to produce antibody:
Prepare Chinese hamster ovary celI, and estimate the ADCC activity of the antibody of this cell generation, the genome area that contains Chinese hamster ovary celI FUT8 gene extron 2 in the said Chinese hamster ovary celI is deleted.
1. make up Chinese hamster FUT8 gene extron 2 targeting vector plasmid pKOFUT8Puro
(1) makes up plasmid ploxPPuro
Plasmid ploxPPuro makes up (Fig. 4) according to the following steps.
Dissolving 1.0 μ gpKOSelectPuro (producing) in 35 μ l NE damping fluids 4 (producing) by Lexicon by New England Biolabs; To wherein adding 20 unit limit property enzyme AscI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the purine-containing mycin resistant gene ceneme of the about 1.5Kb of purifying.
On the other hand; The plasmid ploxP 1.0 μ g that in day not unexamined patent of the present disclosure application No.314512/99, describe are dissolved in the 35 μ l NE damping fluids 4 (being produced by New England Biolabs); To wherein adding 20 unit limit property enzyme AscI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 2.0Kb of purifying.
Mix (the 4.5 μ l of the AscI-AscI fragment derived from plasmid pKOSelectPuro that obtain; About 1.5Kb), 0.5 μ l is derived from the AscI-AscI fragment (approximately 2.0Kb) and 5.0 μ lLigation High (being produced by Toyobo) of plasmid ploxP, 16 ℃ of combinations subsequently 30 minutes.Through using reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called ploxPPuro.
(2) make up plasmid pKOFUT8gE2-1
Adopt the plasmid pFUT8fgE2-2 that obtains in the reference implementation example (2), make up plasmid pKOFUT8gE2-1 through following steps, plasmid pFUT8fgE2-2 has the genome area (Fig. 5) that contains Chinese hamster FUT8 exon 2.
Dissolving 2.0 μ g plasmid pFUT8fgE2-2 in the 35 μ l NE damping fluids 1 that contain 100 μ g/ml BSA (New England Biolabs production) (New England Biolabs production); To wherein adding 20 unit limit property enzyme SacI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 35 μ l NE damping fluids 2 (New England Biolabs production) that contain 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme EcoRV (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 1.5Kb of purifying.
Respectively; 1.0 being dissolved in 35 μ l, μ g plasmid LITMUS28 (New England Biolabs production) contains in the NE damping fluid 1 (New England Biolabs production) of 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme Sad (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 35 μ l NE damping fluids 2 (New England Biolabs production) that contain 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme EcoKV (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 2.8Kb of purifying.
Mix (the 4.5 μ l of the EcoRV-SacI fragment derived from plasmid pFUT8fgE2-2 that obtain; About 1.5Kb), 0.5 μ l is derived from the EcoKV-SacI fragment (approximately 2.8Kb) and 5.0 μ l Ligation High (being produced by Toyobo) of plasmid LITMUS28,16 ℃ of combinations subsequently 30 minutes.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pKOFUT8gE2-1.
(3) make up plasmid pKOFUT8gE2-2
Adopt the plasmid pKOFUT8gE2-1 that obtains in (2) item, make up plasmid pKOFUT8gE2-2 (Fig. 6) through following steps.
Dissolving 2.0 μ g plasmid pKOFUT8gE2-1 in the 30 μ l NE damping fluids 2 that contain 100 μ g/ml BSA (New England Biolabs production) (New England Biolabs production); To wherein adding 20 unit limit property enzyme EcoRV (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 30 μ l NE damping fluids 1 (New England Biolabs production) that contain 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme KpnI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 1.5Kb of purifying.
Respectively, 1.0 μ g plasmid ploxPPuro are dissolved in the 30 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 20 unit limit property enzyme HpaI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 30 μ l NE damping fluids 1 (New England Biolabs production) that contain 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme KpnI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 3.5Kb of purifying.
Derived from behind the HpaI-KpnI fragment (approximately 3.5Kb) of plasmid ploxPPuro and the 5.0 μ l Ligation High (by Toyobo production), mixture carried out association reaction 30 minutes at 16 ℃ to the 4.0 μ l that mix to obtain derived from the EcoKV-KpnI fragment (approximately 1.5Kb) of plasmid pKOFUT8gE2-1,1.0 μ l.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pKOFUT8gE2-2.
(4) make up plasmid pscFUT8gE2-3
Adopt the plasmid pFUT8fgE2-4 that obtains in the reference implementation example (2), make up plasmid pscFUT8gE2-3 through following steps, plasmid pFUT8fgE2-4 has the genome area (Fig. 7) that contains Chinese hamster FUT8 exon 2.
Dissolving 2.0 μ g plasmid pFUT8fgE2-4 in 35 μ l NE damping fluids 1 (New England Biolabs production), to wherein adding 20 unit limit property enzyme HpaII (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation, use Blunting High (Toyobo production) the DNA end to be changed over blunt ends then according to producer's working instructions.Reclaim dna fragmentation through benzene/chloroform extraction and ethanol sedimentation; And be dissolved in the 35 μ l NE damping fluids 2 (New England Biolabs production); To wherein adding 20 unit limit property enzyme HindIII (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 3.5Kb of purifying.
Respectively; 1.0 μ g plasmid LITMUS39 (New England Biolabs production) is dissolved in the 35 μ l NE damping fluids 2 (New England Biolabs production); Solution mixes with 20 unit limit property enzyme EcoRV (New England Biolabs production) and 20 unit limit property enzyme HindIII (New England Biolabs production), and 37 ℃ of digestion 2 hours.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 2.8Kb of purifying.
Mix (the 4.0 μ l of the HpaII-HindIII fragment derived from plasmid pFUT8fgE2-4 that obtain; About 3.5Kb), 1.0 μ l are derived from the EcoRV-HindIII fragment (approximately 2.8Kb) and 5.0 μ l Ligation High (being produced by Toyobo) of plasmid LITMUS39,16 ℃ of combinations subsequently 30 minutes.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pscFUT8gE2-3.
(5) make up plasmid pKOFUT8gE2-3
Adopt the plasmid pFUT8fgE2-4 that obtains in the reference implementation example (2), make up plasmid pKOFUT8gE2-3 through following steps, plasmid pFUT8fgE2-4 has the genome area (Fig. 8) that contains Chinese hamster FUT8 exon 2.
Dissolving 2.0 μ g plasmid pFUT8fgE2-4 in the NE damping fluid (New England Biolabs production) of 35 μ l EcoRI; To wherein adding 20 unit limit property enzyme EcoRI (being produced by New England Biolabs) and 20 unit limit property enzyme HindIII (New England Biolabs production), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 1.8Kb of purifying.
Respectively; 1.0 μ g plasmid pBluescript II KS (+) (Stratagene production) is dissolved in the NE damping fluid (New England Biolabs production) of 35 μ lEcoRI; To wherein adding 20 unit limit property enzyme EcoRI (New England Biolabs production) and 20 unit limit property enzyme HindIII (New England Biolabs production), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 3.0Kb of purifying.
Mix (the 4.0 μ l of the Hindlll-EcdRI fragment derived from plasmid pFUT8fgE2-4 that obtain; About 1.8Kb), 1.0 μ l are derived from the HindIII-EcoRI fragment (approximately 3.0Kb) and 5.0 μ l Ligation High (being produced by Toyobo) of plasmid pBluescript II KS (+), 16 ℃ of combinations subsequently 30 minutes.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pKOFUT8gE2-3.
(6) make up plasmid pKOFUT8gE2-4
Adopt the plasmid pscFUT8fgE2-3 and the pKOFUT8gE2-3 that obtain in (4) item and (5) item, make up plasmid pKOFUT8gE2-4 (Fig. 9) through following steps.
Contain dissolving 1.0 μ g plasmid pscFUT8gE2-3 in the SalI NE damping fluid (New England Biolabs production) of 100 μ g/ml BSA (New England Biolabs production) at 35 μ l; To wherein adding 20 unit limit property enzyme SalI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in 30 μ l and contain in the NE damping fluid 2 (New England Biolabs production) of 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme HindIII (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 3.6Kb of purifying.
Respectively; 1.0 μ g plasmid pKOFUT8gE2-3 (New England Biolabs production) is dissolved in the NE damping fluid (New England Biolabs production) of 35 μ l SalI; To wherein adding 20 unit limit property enzyme SalI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 35 μ l NE damping fluids 2 (New England Biolabs production); To wherein adding 20 unit limit property enzyme HindIII (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion,, then reacted 30 minutes so that the terminal dephosphorylation of DNA at 65 ℃ to wherein adding 35 μ l 1mol/l Tris-HCl damping fluids (pH 8.0) and 3.5 μ l intestinal bacteria CIS deutero-SEAPs (Takara Shuzo production).After dephosphorylation is handled, reclaim dna fragmentation through benzene/chloroform extraction and ethanol sedimentation, and be dissolved in the 10 μ l sterilized waters.
Mix (the 4.0 μ l of the SalI-HindIII fragment derived from plasmid pscFUT8gE2-3 that obtain; About 3.1Kb), 1.0 μ l are derived from the Sall-HindIII fragment (approximately 4.8Kb) and 5.0 μ l Ligation High (being produced by Toyobo) of plasmid pKOFUT8gE2-3,16 ℃ of combinations subsequently 30 minutes.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pKOFUT8gE2-4.
(7) make up plasmid pKOFUT8gE2-5
Adopt the plasmid pKOFUT8gE2-2 and the pKOFUT8gE2-4 that obtain in (3) item and (6) item, make up plasmid pKOFUT8gE2-5 (Figure 10) through following steps.
Dissolving 1.0 μ g plasmid pKOFUT8gE2-2 in 30 μ l NE damping fluids 4 (New England Biolabs production), to wherein adding 20 unit limit property enzyme Smal (being produced by New England Biolabs), 25 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 30 μ l NE damping fluids 2 (New England Biolabs production); To wherein adding 20 unit limit property enzyme BamHI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion,, then reacted 1 hour so that the terminal dephosphorylation of DNA at 65 ℃ to wherein adding 30 μ l 1mol/l Tris-HCl damping fluids (pH 8.0) and 3.0 μ l intestinal bacteria C15 deutero-SEAPs (Takara Shuzo production).After dephosphorylation is handled, reclaim dna fragmentation through benzene/chloroform extraction and ethanol sedimentation, and be dissolved in the 10 μ l sterilized waters.
Respectively, 1.0 μ g plasmid pKOFUT8gE2-4 are dissolved in the 30 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 20 unit limit property enzyme SmaI (New England Biolabs production), 25 ℃ digested 2 hours subsequently.From reaction soln, reclaim dna fragmentation through ethanol sedimentation; And be dissolved in the 30 μ l NE damping fluids 2 (New England Biolabs production); To wherein adding 20 unit limit property enzyme BamHI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out the dna fragmentation of 0.8% (w/v) agarose gel electrophoresis with the about 5.2Kb of purifying.
Mix (the 0.5 μ l of the SmaI-BamHI fragment derived from plasmid pKOFUT8gE2-2 that obtains; About 5.0Kb), 4.5 μ l are derived from the Smal-BamH1 fragment (approximately 5.4Kb) and 5.0 μ l Ligation High (being produced by Toyobo) of plasmid pKOFUT8gE2-4,16 ℃ of combinations subsequently 15 hours.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pKOFUT8gE2-5.
(8) make up plasmid pKOFUT8Puro
Adopt the plasmid pKOFUT8gE2-5 that obtains in (7) item, make up plasmid pKOFUT8Puro (Figure 11) through following steps.
Dissolving 1.0 μ g plasmid pKOSelectDT (producing) in 50 μ l NE damping fluids 4 (New England Biolabs production) by Lexicon; To wherein adding 16 unit limit property enzyme RsrII (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion, solution carries out 0.8% (w/v) agarose gel electrophoresis contains the diphtheria toxin ceneme with the about 1.2Kb of purifying dna fragmentation.
Respectively, 1.0 μ g plasmid pKOFUT8gE2-5 are dissolved in the 50 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 16 unit limit property enzyme RsrII (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion,, then reacted 1 hour so that the terminal dephosphorylation of DNA at 65 ℃ to wherein adding 30 μ l1mol/l Tris-HCl damping fluids (pH 8.0) and 3.0 μ l intestinal bacteria CIS deutero-SEAPs (Takara Shuzo production).After dephosphorylation is handled, reclaim dna fragmentation through benzene/chloroform extraction and ethanol sedimentation, and be dissolved in the 10 μ l sterilized waters.
Mix (the 1.0 μ l of the RsrII-RsrII fragment derived from plasmid pKOSelectDT that obtain; About 1.2Kb), 1.0 μ l are derived from RsrII-RsrII fragment (approximately 10.4Kb), 3.0 μ l sterilized waters and the 5.0 μ l Ligation High (being produced by Toyobo) of plasmid pKOFUT8gE2-5,16 ℃ combine 30 minutes subsequently.With reaction soln transformed into escherichia coli DH5 α strain, isolated plasmid dna from the Ampicillin Trihydrate resistance clone that obtains in accordance with known methods.At this, plasmid is called pKOFUT8Puro.
2. preparation Chinese hamster ovary celI, a copy that wherein contains the genome area of FUT8 gene extron 2 ruptures
(1) imports targeting vector
The Chinese hamster FUT8 genome area targeting vector pKOFUT8Puro that in 1 of this embodiment, makes up is imported into the clone 5-03 of preparation in (2) of embodiment 1.
Be described below, according to electroporation with the gene of plasmid pKOFUT8Puro import clone 5-03 [Cytofechnology, 3, 133 (1990)].At first; 150 μ g plasmid pKOFUT8Puro are dissolved in the NE damping fluid (New England Biolabs production) of 1.8ml SalI; To wherein adding 600 unit limit property enzyme SalI (New England Biolabs production), 37 ℃ digest 5 hours to obtain linear fragment subsequently.With benzene/chloroform extracting abstraction reaction solution, ethanol sedimentation subsequently, the linear plasmid of recovery is made into the aqueous solution of 1 μ g/ μ l.Respectively, clone 5-03 is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/lKH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after 4 μ l (4 μ g) linear plasmid mixes; All cell-DNA the mixture of volume is transferred into Gene Pulser Cuvette (interelectrode distance 2mm) (being produced by BIO-RAD), uses cytogamy equipment Gene Pulser (being produced by BIO-RAD) to carry out electroporation with 350V pulse pressure and 250 μ F electric capacity then.After carrying out electroporation in an identical manner with 30 test tubes; Cell suspending liquid is suspended in the IMDM substratum (being produced by Life Technologies) that has added 10% foetal calf serum (being produced by Life Technologies) and 1 * concentration HT additive (Life Technologies production), and inoculation is advanced in the adherent cell incubator (Falcon production) of 30 10cm diameters.At 5%CO 2In 37 ℃ cultivate after 24 hours, remove the culture supernatant thing, give 10ml and added the dialyse IMDM substratum (Life Technologies production) of serum (Life Technologies production) of 15 μ g/ml tetracyclines (SIGMA productions) and 10% tire ox.Whenever repeated to change culture medium culturing after 10 days, obtained tetracycline-resistance clone at a distance from 3 to 4 days.
(2) preparation has imported the clone of targeting vector
Obtain any 900 colonies in the tetracycline-resistance clone that obtains in (1) item as follows.
At first, from the 10cm dish that has formed tetracycline-resistance clone colony, remove the culture supernatant thing, and placing the dish under the stereoscopic microscope to add the 7ml phosphate buffered saline buffer afterwards.Then, scrape each colony, draw and shift in the 96 hole circle base plates (Falcon production) with Pipetteman (producing) by GILSON.After the trypsin treatment; Each clone is advanced the flat flat board in 96 holes by inoculation and is used to attach cell cultures (Iwaki Glass production), in the IMDM substratum (Life Technologies production) that has added 15 μ g/ml tetracyclines (SIGMA production) and 10% tire ox dialysis serum (Life Technologies production), cultivates for 1 week.
After the cultivation, each clone in the flat board is by trypsin treatment, and (20%DMSO, 40% foetal calf serum 40%IMDM) mix with the freezing substratum of two volumes then.One semifused is inoculated in the flat flat board in 96 holes (Iwaki Glass production) of into attaching cell cultures as copy board, and a remaining semifused by low temperature storage as motherboard.In the IMDM substratum (Life Technologies production) that has added 15 μ g/ml tetracyclines (SIGMA production) and 10% tire ox dialysis serum (Life Technologies production), cultivate 1 week of copy board.
(3) through genome PCR diagnosis homologous recombination
Carry out 900 clones' of acquisition in (2) item homologous recombination diagnosis through genome PCR.
At first, in accordance with known methods [Analytical Biochemistry, 201,331 (1992)] each cloned genes group DNA in the copy board for preparing in preparation (2) item, and dissolving is spent the night in 30 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA, 200 μ g/ml ribonuclease As).In addition, in design and the reference implementation scheme between the FUT8 genome area that obtains the outer sequence bonded primer of targeting vector homologous region (describing) by SEQ ID NO:15 and with carrier in loxP sequence bonded primer (by SEQ ID NO:16 description).
With archaeal dna polymerase ExTaq (Takara Shuzo production); Preparation contains 25 μ l reaction solns [the ExTaq damping fluid (Takara Shuzo production) of each genomic dna solution of 10 μ l above-prepared; 0.2mmol/l dNTPs and 0.5 μ mol/l gene specific primer (SEQ ID NOs:15 and 16)], and carry out polymerase chain reaction (PCR).Through 94 ℃ of heating 3 minutes, 94 ℃ 1 minute, 60 ℃ of 38 round-robin heating subsequently 1 minute and 72 ℃ 2 minutes carried out PCR as a circulation.
Behind the PCR, reaction soln carries out 0.8% (w/v) agarose gel electrophoresis, and approximately the specific amplification fragment that contains marginarium between Chinese hamster ovary celI genome district and the targeting vector homologous region of 1.7Kb is confirmed as positive colony.Find a positive colony with this method.
(4) through genome Southern trace diagnosis homologous recombination
A positive signal clone to confirming in (3) item carries out the homologous recombination diagnosis through genome Southern trace.
In (2) in the middle of the motherboard of freezing preservation, it is dull and stereotyped and at 5%CO to be chosen in 96 holes of finding to contain positive colony in (3) 2Hatched 10 minutes for following 37 ℃.After hatching, from advancing to attach in the 24 hole flat undersides (Greiner production) of cell corresponding to collecting cell the hole of positive colony and inoculation.After 1 week of cultivation, cell is inoculated in the 6 hole flat undersides of attaching cell (Greiner production) in the IMDM substratum (Life Technologies production) that has added 15 μ g/ml tetracyclines (SIGMA production) and 10% tire ox dialysis serum (Life Technologies productions).[Nucleic Acids Research in accordance with known methods; 3; 2303 (1976)] each cloned genes group DNA of preparation from flat board; And dissolving is spent the night in 150 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA, 200 μ g/ml ribonuclease As).
The genomic dna that dissolving 12 μ g obtain in 120 μ l NE damping fluids 3 (New England Biolabs production), to wherein adding 25 unit limit property enzyme PstI (New England Biolabs production), 37 ℃ of digestion are subsequently spent the night.From reaction soln, reclaim dna fragmentation through ethanol sedimentation, be dissolved in the 20 μ l TE damping fluids (pH 8.0,10mmol/l Tris-HCl, lmmol/l EDTA), carry out 0.8% (w/v) agarose gel electrophoresis then.Behind the electrophoresis, genomic dna [Proc.Natl.Acad.Sci.USA, 76,3683 (1979)] is in accordance with known methods transferred on the nylon membrane.After shifting completion, nylon membrane was 80 ℃ of heating 2 hours.
Respectively, the probe preparation of using in the Southern trace as follows.At first, design and the outer sequence bonded primer (SEQ ED NOs:9 and 10) of targeting vector homologous region, the FUT8 genome district homology that obtains in this homologous region and the reference implementation example (2).Then; With archaeal dna polymerase ExTaq (Takara Shuzo production); Preparation contains 20 μ l reaction solns [the ExTaq damping fluid (Takara Shuzo production) of the plasmid pFUT8fgE2-2 that obtains in the 4.0ng reference implementation example (2); 0.2mmol/l dNTPs and 0.5 μ mol/l gene specific primer (SEQ ID NOs:9 and 10) carry out polymerase chain reaction (PCR).Through 94 ℃ of heating 1 minute, 94 ℃ 30 seconds, 55 ℃ of 25 round-robin heating subsequently 30 seconds and 74 ℃ 1 minute carried out PCR as a circulation.Behind the PCR, reaction soln carries out the probe dna fragment of 1.75% (w/v) agarose gel electrophoresis with the about 230bp of purifying.The dna probe solution (5 μ l) that obtains is by labelled with radioisotope, adopt 1.75MBq [α- 32P] dCTP and Megaprime dna marker system, dCTP (Ainersham Pharmacia Biotech production).
Hybridization is carried out as follows.At first, nylon membrane is sealed in and rolls in the bottle, adds 65 ℃ of 15ml hybridization solutions [5 * SSPE, 50 * Denhaldt ' s solution, 0.5% (w/v) SDS, 100 μ g/ml salmon sperm DNAs] and carries out prehybridization in 3 hours.Then, 32The dna probe of P-mark is by thermally denature and put into bottle.Then, spend the night at 65 ℃ of heating nylon membranes.
After the hybridization, in 50ml 2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ the heating 15 minutes.After repeating twice of rinse step, in 50ml 0.2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ of heating 15 minutes.After the flushing, nylon membrane contacted for two nights to develop at-80 ℃ with X line film.
Handle through Restriction Enzyme PstI, from wild-type FUT8 allelotrope, form the dna fragmentation of about 4.4Kb.On the other hand, from allelotrope, formed the dna fragmentation of about 6.0Kb, produced therein with the targeting vector homologous and recombinated.
Through this method, found the specific fragment of this about 4.4Kb and about 6.0Kb in the positive colony genomic dna from (3) item.Because two number of fragments ratios are 1: 1, confirm that this clone is the clone that the allelic copy of FUT8 has ruptured.After this, this clone is called clone Ist.FUTS 2-46.
3. from Chinese hamster ovary celI, delete drug resistant gene, the FUT8 gene copy ruptures in the said Chinese hamster ovary celI
(1) imports Cre recombinase expression vector
Cre recombinase expression vector pBS185 (Life Technologies production) is imported among the clone Ist.FUTS 2-46 of 2 preparations of this embodiment.
Be described below, plasmid pBS185 imported clone Ist.FUT8 2-46 according to electroporation [Cytolechnology, 1,133 (1990)].At first, clone Ist.FUTS 2-46 is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after 4 μ g plasmid pBS185 mixes; All cell-DNA the mixture of volume is transferred into Gene Pulser Cuvette (interelectrode distance 2mm) (being produced by BIO-RAD), uses cytogamy equipment Gene Pulser (being produced by BIO-RAD) to carry out transgenosis with 350V pulse pressure and 250 μ F electric capacity then.
After the transgenosis; Cell suspending liquid is suspended in the 10mlIMDM substratum (being produced by Life Technologies) that has added 10% foetal calf serum (being produced by Life Technologies) and 1 * concentration HT additive (Life Technologies production); And with 20,000 times of the further dilutions of identical substratum.Cell is inoculated the into adherent cell incubator of 7 10cm diameters (Falcon production), then at 5%CO 2In 37 ℃ cultivated 24 hours.After the cultivation, remove the culture supernatant thing, give the IMDM substratum (Life Technologies production) that 10ml has added 10% tire ox dialysis serum (Life Technologies production).Whenever repeating to change substratum at a distance from 3 to 4 days cultivated 10 days.
(2) preparation has imported the clone of Cre recombinase expression vector
Obtain any 400 colonies among the clone who obtains in (1) item as follows.
At first, from the 10cm dish, remove the culture supernatant thing, and placing the dish under the stereoscopic microscope to add the 7ml phosphate buffered saline buffer afterwards.Then, scrape each colony, draw and shift in the 96 hole circle base plates (Falcon production) with Pipetteman (producing) by GILSON.After the trypsin treatment; Each clone is advanced to attach in the flat flat board in 96 holes (Iwaki Glass production) of cell cultures by inoculation, in the IMDM substratum (Life Technologies production) that has added 10% tire ox dialysis serum (Life Technologies production), cultivates for 1 week.
After the cultivation, with each clone in the trypsin treatment flat board, (20%DMSO, 40% foetal calf serum 40%IMDM) mix with the freezing substratum of two volumes then.One semifused is inoculated in the flat flat board in 96 holes (Iwaki Glass production) of into attaching cell cultures with the preparation copy board, and remaining half by low temperature storage as motherboard.
Then, in the IMDM substratum (Life Technologies production) that has added 15 μ g/ml tetracyclines (SIGMA production) and 10% tire ox dialysis serum (Life Technologies production), cultivated copy board 6 days.Positive colony disappearance under the tetracycline situation is being arranged, and owing to the expression of Cre recombinase, the tetracycline-resistant gene that inserts between the loxP sequence is deleted in the positive colony.Through system of selection, 91 positive colonies have been found.
(3) through the deletion of genome Southern trace diagnosis drug resistant gene
Random 6 clones in the positive colony of finding in (2) item are carried out the deletion of genome Southern trace diagnosis drug resistant gene.
In (2) in the middle of the motherboard of freezing preservation, it is dull and stereotyped and at 5%CO to select to contain 96 holes of 6 positive colonies 2Hatched 10 minutes for following 37 ℃.After hatching, from also inoculating the 24 hole flat undersides (Greiner production) that advance to be used for to attach cell corresponding to collecting cell the hole of each positive colony.After in the IMDM substratum (Life Technologies production) that has added 10% tire ox dialysis serum (Life Technologies production), cultivating for 1 week, cell is inoculated the 6 hole flat undersides (Greiner production) that into are used for attaching cell.[Nucleic Acids Research in accordance with known methods; 3; 2303 (1976)] each cloned genes group DNA of preparation from flat board; And dissolving is spent the night in 150 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, lmmol/l EDTA, 200 μ g/ml ribonuclease As).
The genomic dna that dissolving 12 μ g obtain in the NE damping fluid (New England Biolabs production) of 120 μ l BamHI, to wherein adding 20 unit limit property enzyme BamUI (New England Biolabs production), 37 ℃ of digestion are subsequently spent the night.From reaction soln, reclaim dna fragmentation through ethanol sedimentation, be dissolved in 20 μ l TE damping fluids (pH 8.0,10mmol/l Tris-HCl, lmmol/lEDTA) in, carry out 0.4% (w/v) agarose gel electrophoresis then.Behind the electrophoresis, genomic dna in accordance with known methods [Proc.Natl.Acad.Sci.USA, 76, 3683 (1979)] transfer on the nylon membrane.After shifting completion, nylon membrane was 80 ℃ of heating 2 hours.
Respectively, the probe preparation of using in the Southern trace as follows.At first, the outer sequence bonded primer (SEQ ED NOs:9 and 10) of target carrier homologous region in the FUT8 genome district that obtains in design and the reference implementation scheme.Then; With archaeal dna polymerase ExTaq (Takara Shuzo production); Through preparing reaction soln [the ExTaq damping fluid (Takara Shuzo production) that 20 μ l contain the plasmid pFUT8fgE2-2 that obtains in the 4.0ng reference implementation example (2); 0.2mmol/l dNTPs and 0.5 μ mol/l gene specific primer (SEQ ID NOs:9 and 10) carry out polymerase chain reaction (PCR).Through 94 ℃ of heating 1 minute, 94 ℃ 30 seconds, 55 ℃ of 25 round-robin heating subsequently 30 seconds and 74 ℃ 1 minute carried out PCR as a circulation.Behind the PCR, reaction soln carries out the probe dna fragment of 1.75% (w/v) agarose gel electrophoresis with the about 230bp of purifying.Employing 1.75MBq [α- 32P] dCTP and Megaprime dna marker system, dCTP (Ainersham Pharmacia Biotech production), the dna probe solution that 5 μ l obtain is by labelled with radioisotope.
Hybridization is carried out as follows.At first, nylon membrane is sealed in and rolls in the bottle, adds 65 ℃ of 15ml hybridization solutions [5 * SSPE, 50 * Denhaldt ' s solution, 0.5% (w/v) SDS, 100 μ g/ml salmon sperm DNAs] and carries out prehybridization in 3 hours.Then, 32The dna probe of P-mark is by thermally denature and put into bottle, spends the night at 65 ℃ of heating nylon membranes.
After the hybridization, in 50ml 2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ the heating 15 minutes.After repeating twice of rinse step, in 50ml 0.2 * SSC-0.1% (w/v) SDS, soak film and 65 ℃ of heating 15 minutes.After the nylon membrane flushing, contacted for two nights to develop with X line film at-80 ℃.
Handle through above-described Restriction Enzyme BamHI, from wild-type FUT8 allelotrope, formed the dna fragmentation of about 25.5Kb.And, from allelotrope, formed the dna fragmentation of about 20.0Kb, produced therein with the targeting vector homologous and recombinated.In addition, when tetracycline-resistant gene (approximately 1.5Kb) is deleted, form the dna fragmentation of about 18.5Kb with identical processing from the allelotrope that has produced homologous recombination.
Through this method, from 5 cloned genomic dnas of 6 clones, found the specific fragment of about 25.5Kb and about 18.5Kb.Because two number of fragments ratios are 1: 1, show that tetracycline-resistant gene is deleted from the clone, a copy in this clone FUT8 genome district ruptures.After this, this clone is called clone Ist.FUTS 2-46-1.Equally, the result of clone Ist.FUT8 2-46-1, clone Ist.FUT8 2-46 and clone 5-03 genome Southern trace is presented at Figure 12.And; Clone Ist.FUT8 2-46-1; With the name of 2-46-1 in September 26 calendar year 2001 at International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6,1; Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan) be FERMBP-7755 by preservation.
4. purifying FUT8 gene break is cloned the antibody that produces
The clone Ist.FUT8 2-46-1 that in 3 of present embodiments, obtains through a fracture FUT8 allelic copy is suspended in and has added 15 μ g/ml tetracyclines (SIGMA productions) and 10% tire ox and dialyse in the IMDM substratum (Life Technologies production) of serum (Life Technologies production) to 3 * 10 5Cell/ml density, the 60ml of total then suspension-s are inoculated into, and two T182 flasks are used to attach cell cultures (Greiner production).Cultivate after 3 days, abandon the supernatant thing and change into the EXCELL301 substratum of 60ml (producing) altogether by JRH Biosciences.
At 5%CO 2Cultivate after 7 days for 37 ℃ in the incubator, calculate the intact cell number to confirm that its viability almost is identical (each 30% or following), reclaims each cell suspending liquid then.With 3,4 ℃ of eccentric visual cell suspension-s of 000rpm 10 minutes, with 10, the supernatant thing of 4 ℃ of centrifugal recovery of 000rpm 1 hour filters with the 150ml capacity PES Filter Unit (NALGENE production) with 0.22 μ m aperture then.
Filling Prosep-A High Capacity (bioPROCESSING production) washes to reach the balance carrier with 10ml 0.1mol/l citrate buffer (pH 3.0) and 10ml1mol/l Padil/NaOH-0.15mol/l NaCl damping fluid (pH 8.6) to 2cm thickness in this order in the post of 0.8cm diameter.Then, each culture supernatant thing 100ml is through this post and with 50ml 1mol/l Padil/NaOH-0.15mol/l NaCl damping fluid (pH 8.6) flushing.After the flushing, the antibody of Prosep-A absorption is with 2.5ml 0.1mol/l citrate buffer (pH 3.0) wash-out, and eluate is with 500 μ l fractionation, and each cut is neutralized through mixing with 100 μ l 2mol/l Tris-HCl (pH 8.5).Through the BCA method [Anal.Biochem., 150, 76 (1985)] and select two cuts (1.2ml altogether) that contain high concentration antibody, merge, use 10mol/l Citrate trianion-0.15mol/l NaCl damping fluid (pH 6.0) dialysis diel then.After the dialysis, reclaim antibody-solutions and filtration carries out disinfection with the MillexGV (MILLIPORE production) in 0.22 μ m aperture.
5.FUT8 the ADCC of the antibody compositions that the gene break clone produces is active
Active for the ADCC that estimates the anti-CCR4 antibody of purifying in 4 of this embodiments, it is active to adopt the CCR4-positive colony CCR4/EL-4 that describes among the WO 01/34754 to detect ADCC.
The RPMI1640 substratum that has added 10% foetal calf serum (Life Technologies production) (producing) by Life Technologies go down to posterity in (after this being called " RPMI1640-FBS (10) ") cultivate 1 * 10 6Cell CCR4/EL-4 clone is suspended among the 500 μ l RPMI1640-FBS (10), after this to the Na that wherein adds 3.7MBq 2 51CrO 4, cultivate 90 minutes to use the labelled with radioisotope cell for 37 ℃ subsequently.With 1,200rpm abandons the supernatant thing after centrifugal 5 minutes, and target cell suspension is in 5ml RPMI1640-FBS (10).Repeat 3 times rinse step, then incubated cell suspension-s on ice 30 minutes with spontaneous decomposition radioactivity material.Repeat rinse step again twice, then at 5ml RPMI1640-FBS (1O) thus in suspension cell prepare 2.0 * 10 5The target cell suspension liquid of cell/ml.
Respectively, gather 30ml venous blood, mix gently, mix with 30ml physiology salt (Otsuka Pharmaceutical production) then with 0.5ml heparin sodium (producing) by Shimizu Pharmaceutical from healthy donors.After the mixing, the 10ml mixture covers gently on 4ml Lymphoprep (NYCOMED PHARMA AS production), with 2, and centrifugal 30 minutes of 000rpm room temperature.From centrifuge tube, collect isolating monocyte part, merge, be suspended in then among the 30ml RPMI1640-FBS (10).With 1, the 200rpm room temperature abandons the supernatant thing after centrifugal 15 minutes, and in 20mlRPMI1640-FBS (10) suspension cell.Repeat rinse step twice, use RPMI1640-FBS (10) preparation 2.5 * 10 then 6Effector cell's suspension-s of cell/ml.
With target cell suspension liquid with 50 μ l (1 * 10 4Cells/well) divides to go in each hole of 96 hole U-shaped base plates (Falcon production).Subsequently, effector cell's suspension-s is with 100 μ l (2.5 * 10 5Cells/well) divide to go into every hole, thereby the ratio of regulating effector cell and target cell is 25: 1.Then; The serial dilution solution of each anti-CCR4 Antibody Preparation 0.01 μ g/ml, 0.1 μ g/ml, 1 μ g/ml and the 10 μ g/ml that from 4 of this embodiments, obtain with RPMI1640-FBS (10), the solution of dilution divide in the hand-hole to final concentration with 50 μ l and are respectively 0.0025 μ g/ml, 0.025 μ g/ml, 0.25 μ g/ml and 2.5 μ g/ml.At 5%CO 2In 37 ℃ the reaction 4 hours after, with 1, centrifugal dull and stereotyped 5 minutes of 200rpm.The supernatant thing that in batches takes out 75 μ l in every hole in 12mm diameter RIA pipe (IWAKI productions), with MINAX-γ automatic-gamma counter 5550 (PACKARD production) measurement is dissociated 51The amount of Cr.
In addition, not having to carry out identical reaction in the reaction mixture of effector cell's suspension-s and antibody-solutions adding 150 μ l RPMI 1640-FBS (1O) calculates spontaneous dissociated 51The amount of Cr.It is always dissociated in adding 100 μ l 1N hydrochloric acid and do not have with 50 μ l RPMI1640-FBS (10) reaction mixture of effector cell's suspension-s and antibody-solutions, to carry out identical reaction calculating 51The amount of Cr.
Adopt these values, it is active to calculate ADCC according to the equation of describing in (3) item (I).
Figure 13 shows that the ADCC of each anti-CCR4 antibody is active.The antibody that the antibody that from the clone 1st.FUT8 2-46-1 of a copy fracture of FUT8 allelotrope, the obtains clone 5-03 more preceding than the fracture of CHO clone gene produces shows that significantly high ADCC is active.In addition, do not observe these antibody antigens and combine active change.Confirm based on this result, can improve the ADCC activity of the antibody of generation through FUT8 allelotrope in the fracture host cell.
Embodiment 4
Prepare high resistance clone from ruined Chinese hamster ovary celI of copy of FUT8 gene:
Generally know that; Two all ruined clones of allelotrope are through culturing cell in substratum; Separate resistance clone then and obtain; An allelotrope of the genomic gene that obtains through homologous recombination technique with targeting vector in the culturing cell is destroyed, and the positive selective agent concentration that in substratum, is used to select to insert the targeting vector clone is raised about 10 times and [controls mice embryonic, laboratory manual; Gene target, practical approach, IRL Press at Oxford University Press (1993) is with ES cell preparation two mutants mouse].
Therefore, with the clone Ist.FUTS 2-46 that obtains among 2 (3) of embodiment 3, according to known method [Molecular and Cellular Biology, 12, 2391 (1992)], the ruined transformant preparation of 2 copies of FUT8 gene is as follows.
(1) preparation high density tetracycline-resistance clone
1 * 10 8The clone Ist.FUTS 2-46 of cell concentration is suspended in the IMDM substratum (Life Technologies production) that has added 10% dialysis foetal calf serum (Life Technologies production); And inoculation is advanced in the 10cm dish vessel (Falcon production) of 20 attaching cell cultures, then at 5%CO 2Condition was cultivated 24 hours for following 37 ℃.After the cultivation, abandon the supernatant thing, distribute the IMDM substratum (Life Technologies production) of the foetal calf serum (Life Technologies production) that has added 150 μ g/ml tetracyclines (SIGMA production) and 10% dialysis with 10ml.Whenever repeated to change this substratum, cultivated 12 days at a distance from 3 to 4 days.
Collect 90 resistance colonies that form according to the following steps.At first, from the 10cm dish, abandon the culture supernatant thing,, then dish is placed under the stereoscopic microscope with the replacement of 7ml phosphate buffered saline buffer.Then, peel off colony, draw and shift 96 hole circle base plates (Falcon production) with Pipetteman (GILSON production).After the trypsin treatment; Each clone's inoculation advances to attach the 96 hole flat undersides (Iwaki Glass production) of cell, in the IMDM substratum (Life Technologies production) of the foetal calf serum (Life Technologies production) that has added 150 μ g/ml tetracyclines (SIGMA production) and 10% dialysis, cultivates for 1 week.
After the cultivation, each clone on the flat board accepts trypsin treatment, and (20%DMSO, 40% foetal calf serum 40%IMDM) mix with the freezing substratum of two volumes.The flat flat board in 96 holes (Iwaki Glass productions) that the long-pending inoculation of one of which halfbody advances to attach cell to be preparing copy board, half remaining volume by low temperature storage as motherboard.In the IMDM substratum (Life Technologies production) of the foetal calf serum (Life Technologies production) that has added 15mg/ml tetracycline (SIGMA production) and 10% dialysis, cultivate 1 week of copy board.
(2) through genome Southern trace diagnosis homologous recombination
According to following steps, all resistances clones that obtain in (1) item are carried out the homologous recombination diagnosis through genome Southern trace.
Behind the copy board of preparation, all clones are advanced to attach the 24 hole flat undersides (Greiner production) of cell by inoculation in trypsin treatment (1) item.All are cloned in middle the cultivation for 1 week of IMDM substratum (Life Technologies production) of the foetal calf serum (Life Technologies production) that has added 150 μ g/ml tetracyclines (SIGMA production) and 10% dialysis, advance to attach the 6 hole flat undersides (Greiner production) of cell with trypsin treatment and inoculation.According to known method [Nucleic Acids Research; 3; 2303 (1976)] each cloned genes group DNA of preparation from flat board; And dissolving is spent the night in 150 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/lEDTA, 200mg/ml ribonuclease A).
The genomic dna of dissolving 12 μ g above-prepared in the NE damping fluid (New England Biolabs production) of 120 μ l BamHI is to wherein adding 25 unit limit property enzyme Bam HI (New England Biolabs production), subsequently 37 ℃ of digestion.From reaction soln, reclaim dna fragmentation through ethanol precipitation, be dissolved in the 20 μ l TE damping fluids (pH 8.0,10mmol/l Tris-HCl, 1mmol/l EDTA), carry out 0.6% (w/v) agarose gel electrophoresis then.Behind the electrophoresis, genomic dna according to known method [Proc.Natl.Acad.Sci.USA, 76, 3683 (1979)] transfer on the nylon membrane.After the transfer, nylon membrane was heat-treated 2 hours at 80 ℃.
On the other hand, the preparation of the probe of Southern trace as follows.Adopt archaeal dna polymerase ExTaq (Takara Shuzo production); Through preparing 20 μ l reaction solns [ExTaq damping fluid (Takara Shuzo production); 0.2mmol/l dNTPs, 0.5 μ mol/l said gene special primer (SEQ ID NOs:9 and 10)] carry out polymerase chain reaction (PCR).Through 94 ℃ of heating 1 minute, 94 ℃ 30 seconds, 55 ℃ of 25 round-robin 30 seconds and 74 ℃ were 1 minute subsequently, carry out PCR as a circulation.Behind the PCR, reaction soln carries out the probe dna fragment of 1.75% (w/v) agarose gel electrophoresis with the about 230bp of purifying.Adopt [the α of 1.75MBq 32P] dCTP and Megaprime dna marker system, dCTP (Ainersham Pharmacia Biotech production), the dna probe solution that 5 μ l obtain is by labelled with radioisotope.
Hybridization is carried out as follows.At first, top nylon membrane is sealed in and rolls in the bottle, adds 15ml hybridization solution [5 * SSPE, 50 * Denhaldt ' s solution, 0.5% (w/v) SDS, 100mg/ml salmon sperm DNA] 65 ℃ and carries out prehybridization 3 hours.Then, 32The dna probe of P-mark is put into bottle and 65 ℃ of heated overnight by thermally denature.
After the hybridization, in 50ml 2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ heating 15 minutes.After repeating twice of this rinse step, in 50ml 0.2 * S SC-0.1% (w/v) SDS, soak this film and 65 ℃ of heating 15 minutes.After the flushing, nylon membrane contacted for two nights to develop at-80 ℃ with X line film.
Handle through top Restriction Enzyme BamHI, from wild-type FUT8 allelotrope, obtained the dna fragmentation of about 25.5Kb.On the other hand, from allelotrope, formed the dna fragmentation of about 20.0Kb, described allelotrope results from through the identical Restriction Enzyme processing and the homologous recombination of targeting vector.
According to this method, the clone (Figure 14) of the homologous recombination district specific fragment of about 14.0Kb above finding only to show.This clone is called as clone 2-46-H10.
Embodiment 5
The CHO/DG44 cell that all existing FUT8 genes all rupture on the preparation genome:
Preparation CHO/DG44 clone, the genome district that wherein contains two allelic translation atg start codons of FUT8 is deleted.
1. make up Chinese hamster FUT8 gene extron 2 targeting vector plasmid pKOFUT8Neo
According to following steps; Make up plasmid pKOFUT8Neo (Figure 15) through replace tetracycline-resistant gene ceneme contained among the targeting vector plasmid pKOFUT8Puro with Xin Meisu-resistant gene ceneme, wherein plasmid pKOFUT8Puro obtains in the paragraph 1 of embodiment 3.
Dissolving 1.0 μ g plasmid pKOSelectNeo (Lexicon production) in 50 μ l NE damping fluids 4 (New England Biolabs production), to wherein adding 16 unit limit property enzyme AscI (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.From reaction soln, collect dna fragmentation with ethanol precipitation; And be dissolved in the 50 μ l NE damping fluids 4 (New England Biolabs production) that contain 100 μ g/ml BSA (New England Biolabs production); To wherein adding 20 unit limit property enzyme ApaLI (being produced by New England Biolabs), 25 ℃ digested 2 hours subsequently.After the digestion, the liquid of acquisition carries out 0.8% (w/v) agarose gel electrophoresis, and approximately the dna fragmentation that contains Xin Meisu-resistant gene ceneme of 1.6Kb is by purifying.
Respectively, 1.0 μ g plasmid pKOFUT8Puro are dissolved in the 50 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 16 unit limit property enzyme Ascl (being produced by New England Biolabs), 37 ℃ digested 2 hours subsequently.After the digestion,, reacted 1 hour so that the terminal dephosphorylation of DNA at 65 ℃ to 1mol/l Tris-HCl damping fluid that wherein adds 30 μ lpH8.0 and 3.0 μ l intestinal bacteria C15 deutero-SEAPs (Takara Shuzo production).Behind the dephosphorylation, carry out benzene/chloroform extraction and ethanol sedimentation, the dna fragmentation of recovery is dissolved in the 10 μ l sterilized waters.
The 1.0 μ l that obtain above mixing derived from the AscI-AscI fragment (approximately 1.6Kb) of plasmid pKOSelectNeo, AscI-AscI fragment (approximately 10.1Kb), 3.0 μ l sterilized waters and the 5.0 μ l Ligation Highs (by Toyobo produce) of 1.0 μ l derived from plasmid pKOFUT8Puro after, carried out association reaction 30 minutes at 16 ℃.With reaction soln transformed into escherichia coli DH5 α strain, from the Ampicillin Trihydrate resistance clone that obtains, separate each DNA according to known method.This plasmid is called pKOFUT8Neo.
2. the CHO/DG44 cell that copy of FUT8 gene ruptures on the preparation genome:
(1) the preparation targeting vector imports the clone
In the CHO/DG44 cell from Chinese hamster ovary that dihydrofolate reductase gene (dhfr) lacks [Somatic Cell and Molecular Genetics, 12, 555 (1986)], import the Chinese hamster FUT8 genome district targeting vector pKOFUT8Neo that makes up in 1 of this embodiment.According to following steps, through electroporation with the gene of plasmid pKOFUT8Neo import the CHO/DG44 cell [Cytotechnology (electroporation), 3, 133 (1990)].At first; 280 μ g plasmid pKOFUT8Neo are dissolved in the NE damping fluid (New England Biolabs production) of the 1.2ml SalI that contains 100 μ g/ml BSA (New England Biolabs production); To wherein adding 400 unit limit property enzyme SalI (New England Biolabs production), carried out linearizing in 5 hours then through 37 ℃ of digestion.With benzene/chloroform extracting abstraction reaction solution, ethanol sedimentation subsequently, the linear plasmid of recovery is made into the aqueous solution of 1 μ g/ μ l.Respectively, the CHO/DG44 cell suspension is at K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after the above-mentioned linearization plasmid of 4 μ l (4 μ g) mixes; Cell-DNA the mixture liquid of whole amounts is transferred into Gene Pulser Cuvette (interelectrode distance 2mm) (being produced by BIO-RAD), and carries out the gene importing with cytogamy equipment Gene Pulser (being produced by BIO-RAD) with 350V pulse pressure and 250 μ F electric capacity.The cell suspending liquid of quiding gene is suspended in the IMDM substratum (being produced by Invitrogen) that has added 10% foetal calf serum (being produced by Invitrogen) and 1 * concentration HT additive (Invitrogen production), and is seeded on the adherent cell incubator (Falcon production) of 10cm.At 5%CO 2In 37 ℃ cultivate after 24 hours; Remove the culture supernatant thing, the IMDM substratum (Invitrogen production) that has added 600 μ g/ml G418 (Nacalai Tesque productions), 1 * concentration TIT additive (Invitrogen production) and 10% foetal calf serum (Invitrogen production) is distributed to each hole with the 10ml/ hole.Whenever repeated to change substratum at a distance from 3 to 4 days and cultivate 15 days to obtain the G418-resistance clone.
(2) through genome PCR diagnosis homologous recombination
According to following steps, (1) G418-resistance clone that obtains is carried out the homologous recombination diagnosis through genome PCR.
With the G418-resistance clone that obtains on the trypsin treatment 96 hole flat boards, (20%DMSO, 40% foetal calf serum 40%IMDM) mix with the freezing substratum of doubling dose then.The miscellany of half amount is inoculated into 96 hole flat undersides of attaching cell (Asahi Technoglass production) as copy board, and remaining half is used for low temperature storage as motherboard.
In the IMDM substratum (Invitrogen production) that has added 600 μ g/ml G418 (Nacalai Tesque productions), 1 * concentration HT additive (Invitrogen production) and 10% foetal calf serum (Invitrogen production), hatch 1 week of copy board; According to known method [AnalyticalBiochemistry 201, 331 (1992)] and prepare each cloned genes group DNA, their each dissolvings in 30 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l ETDA, 200 μ g/ml ribonuclease As) are spent the night.
Thereafter (PCR) carries out as follows in polymerase chain reaction; Use in the FUT8 genome district that obtains with the reference implementation scheme outside the target carrier homologous region sequence bonded primer (SEQ ID NO:13 or 15) of part and with interior carrier sequence bonded primer (SEQ ID NO:14 or 16).Specifically; Prepare 25 μ l reaction solns [archaeal dna polymerase ExTaq (Takara Shuzo production); ExTaq damping fluid (Takara Shuzo production), 0.2mmol/l dNTPs, (forward primer is shown by SEQ ID NO:13 or 15 the special primer of 0.5 μ mol/l said gene; Reverse primer is shown by SEQ ID NO:14 or 16)]; Said reaction soln contains each genomic dna solution 10 μ l of above-prepared, and through 94 ℃ of heating 3 minutes, 94 ℃ of heating 1 minute, 60 ℃ 1 minute and 72 ℃ 2 minutes carried out PCR as a circulation subsequently.
Behind the PCR, reaction soln carries out 0.8% (w/v) agarose gel electrophoresis, and the specific amplified of observed 1.7Kb comprises the boundary member of Chinese hamster ovary celI genome district and targeting vector homologous region, is confirmed as positive colony (50-10-104).
(3) through genome Southern trace diagnosis homologous recombination
According to following steps, the positive that obtains in (2) item is confirmed that the clone carries out the homologous recombination diagnosis through genome Southern trace.
In the middle of the motherboard of freezing state storage, it is dull and stereotyped to be chosen in 96 holes of finding in (2) item that contain positive colony, makes it at 5%CO 2Placed 10 minutes for following 37 ℃.After the placement, from being seeded in corresponding to the cell in the positive colony hole on the 24 hole flat undersides (Greiner production) that attach cell.Adding 600 μ g/ml G418 (producing) by Nacalai Tesque; After cultivating for 1 week in the IMDM substratum (Invitrogen production) of 1 * concentration HT additive (Invitrogen production) and 10% foetal calf serum (Invitrogen production), cell is seeded on the 6 hole flat undersides (Greiner production) that attach cell.[Nucleic Acids Research in accordance with known methods; 3; 2303 (1976)] each cloned genes group DNA of preparation from flat board; And dissolving is spent the night in 150 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA, 200 μ g/ml ribonuclease As).
The genomic dna of dissolving 12 μ g above-prepared in the NE damping fluid (New England Biolabs production) of the 120 μ l BamHI that contain 100 μ g/ml BSA (New England Biolabs production); To wherein adding 25 unit limit property enzyme BamHL (New England Biolabs production), 37 ℃ of digestion are subsequently spent the night.From reaction soln, collect dna fragmentation through ethanol precipitation, be dissolved in the 20 μ l TE damping fluids (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA), and carry out 0.6% (w/v) agarose gel electrophoresis.Behind the electrophoresis, genomic dna [Proc.Natl.Acad.Sci.USA, 76,3683 (1979)] is in accordance with known methods transferred on the nylon membrane.After the transfer, nylon membrane was heat-treated 2 hours at 80 ℃.
Respectively, the preparation of the probe of Southern trace as follows.At first, the sequence bonded primer (SEQ ID NO:9 and 10) according to part outside the target carrier homologous region in the FUT8 genome district of following step use and the acquisition of reference implementation example carries out PCR.Specifically; Prepare 20 μ l reaction solns [archaeal dna polymerase ExTaq (Takara Shuzo production); ExTaq damping fluid (Takara Shuzo production), 0.2mmol/l dNTPs, the primer (SEQ ID NO:9 and 10) that 0.5 μ mol/l said gene is special]; Said reaction soln contains the plasmid pFUT8fgE2-2 that obtains in the 4.0ng reference implementation example (2), carries out PCR through 1 minute, 94 ℃ 30 seconds, 55 ℃ of 94 ℃ of heating of 25 round-robin 30 seconds and 74 ℃ 1 minute as a circulation.Behind the PCR, reaction soln carries out 1.75% (w/v) agarose gel electrophoresis, and approximately the probe dna fragment of 230bp is by purifying.Adopt [α- 32P] dCTP 1.75MBq and Megaprime dna marker system, dCTP (Ainersham Pharmacia Biotech production) carries out labelled with radioisotope to 5 μ l dna probe solution.
Hybridization is carried out as follows.At first, above-mentioned nylon membrane is encapsulated in and rolls in the bottle, to wherein adding 15ml hybridization solution [5 * SSPE, 50 * Denhaldt ' s solution, 0.5% (w/v) SDS, 100 μ g/ml salmon sperm DNAs], subsequently 65 ℃ of hybridization 3 hours.Then, use 32The dna probe of P-mark is by thermally denature and pour in the bottle 65 ℃ of heated overnight into.
After the hybridization, in 50ml 2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ heating 15 minutes.After repeating twice of above-mentioned flushing operation, in 50ml 0.2 * SSC-0.1% (w/v) SDS, soak this film and 65 ℃ of heating 15 minutes.After the flushing, nylon membrane contacts to develop with X line film at-80 ℃.
Handle with Restriction Enzyme BamHI through above-mentioned, from wild-type FUT8 allelotrope, obtained the dna fragmentation of about 25.5Kb.On the other hand, handle, from the allelotrope that the targeting vector homologous recombination takes place, obtained the dna fragmentation of about 20.0Kb through identical Restriction Enzyme.
Based on present method, from the genomic dna of positive colony 50-10-104, found the above-mentioned specific fragment (Figure 16) of about 25.5Kb and about 20.0Kb.Because two number of fragments ratios are 1: 1, confirm that the clone is the clone who partly knocks out, wherein the allelic copy of FUT8 ruptures.
In the preparation genome FUT8 gene by two CHO/DG44 cells that knock out
(1) clone of preparation targeting vector importing
The Chinese hamster FUT8 genome district targeting vector pKOFUT8Puro that in 1 of embodiment 3, makes up is imported into the FUT8 that obtains in 2 of the present embodiments and partly knocks out clone 50-10-104.
According to following steps, through electroporation import plasmid pKOFUT8Puro gene [Cytotechnology (electroporation), 3, 133 (1990)].At first; 440 μ g plasmid pKOFUT8Puro are dissolved in the NE damping fluid (New England Biolabs production) of the 2.4ml SalI that contains 100 μ g/ml BSA (New England Biolabs production); To wherein adding 800 unit limit property enzyme SalI (New England Biolabs production), carried out linearizing in 5 hours through 37 ℃ of digestion.With benzene/chloroform extracting abstraction reaction solution, ethanol sedimentation subsequently, the linear plasmid of recovery is made into the aqueous solution of 1 μ g/ μ l.Respectively, 50-10-104 is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/lMgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after the above-mentioned linearization plasmid of 4 μ l (4 μ g) mixes; Cell-DNA the mixture liquid of whole amounts is transferred into Gene Pulser Cuvette (interelectrode distance 2mm) (being produced by BIO-RAD), and carries out the gene importing with Gene Pulser cytogamy equipment (being produced by BIO-RAD) with 350V pulse pressure and 250 μ F electric capacity.The cell suspending liquid of quiding gene is suspended in the IMDM substratum (being produced by Invitrogen) that has added 10% foetal calf serum (being produced by Invitrogen) and 1 * concentration HT additive (Invitrogen production), and is seeded on the adherent cell culture dish (Falcon production) of 10cm.At 5%CO 2With 37 ℃ of following incubated cells after 24 hours; Remove the culture supernatant thing, with the IMDM substratum (Invitrogen production) of 10ml distribution interpolation the 15 μ g/ml tetracyclines (SIGMA productions), 1x concentration HT additive (Invitrogen production) and 10% foetal calf serum (Invitrogen production).Whenever repeated the substratum replacing at a distance from 7 days and cultivate 15 days to obtain the resistance clone.
(2) through genome Southern trace diagnosis homologous recombination
According to following steps, the resistance clone who obtains in (1) item is carried out the homologous recombination diagnosis through genome Southern trace.
From the 10cm dish of finding tetracycline-resistance clone, remove the culture supernatant thing, inject the 7ml phosphate buffered saline buffer, transfer under the stereoscopic microscope then.Then, scrape colony, draw and collect in the 96 hole circle base plates (Falcon production) with Pipetteman (GILSON production).After the trypsin treatment; Each clone is seeded on the 96 hole flat undersides (Asahi Technoglass production) that attach cell, in the IMDM substratum (Invitrogen production) that has added 15 μ g/ml tetracyclines (SIGMA productions), 1 * concentration HT additive (Invitrogen production) and 10% foetal calf serum (Invitrogen production), cultivates for 1 week.
After the cultivation,, plant then on the 24 hole flat undersides that attach cell (Greiner production) with each dull and stereotyped clone of trypsin treatment.After in the IMDM substratum (Invitrogen production) that has added 15 μ g/ml tetracyclines (SIGMA productions), 1 * concentration HT additive (Invitrogen production) and 10% foetal calf serum (Invitrogen production), cultivating for 1 week, the clone is seeded on the 6 hole flat undersides (Greiner production) that attach cell.According to known method [Nucleic Acids Research, 3, 2303, (1976)] from each cloned genes group DNA of flat board preparation, dissolving is spent the night in 150 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l ETDA, 200 μ g/ml ribonuclease As).
The genomic dna of dissolving 12 μ g above-prepared in the NE damping fluid (New England Biolabs production) of the 120 μ l BamHI that contain 100 μ g/ml BSA (New England Biolabs production); Add 25 unit limit property enzyme BamHI (New England Biolabs production), 37 ℃ of digestion are subsequently spent the night.From reaction soln, collect dna fragmentation through ethanol precipitation, be dissolved in the 20 μ l TE damping fluids (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA), and carry out 0.6% (w/v) agarose gel electrophoresis.Behind the electrophoresis, genomic dna in accordance with known methods [Proc.Natl.Acad.Sci.USA, 76, 3683 (1979)] transfer on the nylon membrane.After the transfer, nylon membrane was heat-treated 2 hours at 80 ℃.
Respectively, the used probe of Southern trace prepares as follows.At first, the sequence bonded primer (SEQ ID NO:11 or 12) of part outside the target carrier homologous region carries out PCR according to following steps in use and the FUT8 genome district.Specifically; Prepare 20 μ l reaction solns [archaeal dna polymerase ExTaq (Takara Shuzo production), ExTaq damping fluid (Takara Shuzo production), 0.2mmol/l dNTPs; 0.5 the gene specific primer (SEQ ID NO:11 and 12) that μ mol/l is above-mentioned]; Said reaction soln contains the plasmid pFUT8fgE2-2 that obtains in the 4.0ng reference implementation example (2), and through 94 ℃ of heating 1 minute, 94 ℃ 30 seconds, 55 ℃ of 25 round-robin 30 seconds and 74 ℃ of reactions in 1 minute were subsequently carried out PCR as a circulation.Behind the PCR, reaction soln carries out 1.75% (w/v) agarose gel electrophoresis, and approximately the probe dna fragment of 230bp is by purifying.Adopt [α- 32P] dCTP1.75MBq and Megaprime dna marker system, dCTP (Amersham Pharmacia Biotech production) carry out radio-labeled to 5 μ l dna probe solution.
Hybridization is carried out as follows.At first, above-mentioned nylon membrane is encapsulated in and rolls in the bottle, to wherein adding 15ml hybridization solution [5 * SSPE, 50 * Denhalt ' s solution, 0.5% (w/v) SDS, 100 μ g/ml salmon sperm DNAs], carries out prehybridization 3 hours at 65 ℃.Then, use 32The dna probe of P mark is by thermally denature and pour in the bottle 65 ℃ of heated overnight into.
After the hybridization, in 50ml 0.2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ heating 15 minutes.Repeat above-mentioned flushing operation twice, in 50ml0.2 * SSC-0.1% (w/v) SDS, soak then this film and 65 ℃ the heating 15 minutes.After the flushing, nylon membrane contacts to develop with X line film at-80 ℃.
Handle with Restriction Enzyme BamHI through above-mentioned, from wild-type FUT8 allelotrope, obtained the dna fragmentation of about 25.5Kb.On the other hand, handle, from the allelotrope that the targeting vector homologous recombination takes place, obtained the dna fragmentation of about 20.0Kb through identical Restriction Enzyme.
Through present method, from the genomic dna of resistance clone WK704, only found the homologous recombination district specific fragment (Figure 17) of about 20.0Kb.Because the fragment special to wild-type allele disappears (arrow indication part), confirm that the clone is the clone of all existing FUT8 allelotrope fracture on the genome.
4. from the CHO/DG44 cell that the FUT8 Gene Double knocks out, remove drug resistant gene
(1) imports Cre recombinase expression vector
Cre recombinase expression vector pBS185 (Life Technologies production) is imported into two the knocking out among clone's the WK704 of FUT8 of 3 preparations of this embodiment.
According to following steps, through electroporation [Cytolechnology, 3, 133 (1990)] and with two the knocking out among the clone of each FUT8 of plasmid pBS185 gene importing.At first, each FUT8 is two knocks out the clone and is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/lNa 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after 4 μ g plasmid pBS185 mixes; All cell-DNA the mixture liquid of amount is transferred into Gene Pulser Cuvette (electrode distance: 2mm) (produced by BIO-RAD), carry out gene with cytogamy equipment Gene Pulser (being produced by BIO-RAD) with 350V pulse pressure and 250 μ F electric capacity and import.After the importing; Each cell suspending liquid is suspended in the IMDM substratum (by Invitrogen production) that 10ml added 10% foetal calf serum (being produced by Invitrogen) and 1 * concentration HT additive (Invitrogen production); And with 20,000 times of the further dilutions of identical substratum.The solution of each dilution is seeded on the adherent cell culture dish (Falcon production) of 7 10cm, at 5%CO 2In 37 ℃ hatch 10 days to form colony.
(2) preparation Cre recombinase expression vector imports the clone
Collect clone arbitrarily according to following steps the colony that obtains from importing by the WK704 gene.At first, from the 10cm dish, remove the culture supernatant thing, inject the 7ml phosphate buffered saline buffer, transfer under the stereoscopic microscope then.Then, scrape colony, draw and be collected in the 96 hole circle base plates (Falcon production) with Pipetman (producing) by GILSON.After the trypsin treatment; Each clone is seeded on the flat flat board in 96 holes (Iwaki Glass production) that attaches cell, in the IMDM substratum (Invitrogen production) that has added 10% foetal calf serum (Invitrogen production) and 1 * concentration HT additive (Invitrogen production), cultivates for 1 week.
After the cultivation, with each clone in the flat board above the trypsin treatment, with freezing substratum (20%DMSO, 40% foetal calf serum, 40%IMDM) mixing of doubling dose.Its half amount is seeded in the 96 hole flat undersides (Asahi Technoglass productions) that attach cell and goes up as copy board, on the other hand, half that be left by low temperature storage as motherboard.
Then, in the IMDM substratum (Invitrogen production) that has added 600 μ g/ml G418 (Nacalai Tesque productions), 15 μ g/ml tetracyclines (SIGMA production), 10% foetal calf serum (Invitrogen production) and 1 * concentration HT additive (Invitrogen production), hatched copy board 7 days.According to the expression of Cre recombinase, having under G418 and the tetracycline situation positive colony disappear, in the positive colony between the loxP sequence drug resistant gene on two allelotrope be removed.With this negative positive colony of having selected scientific discovery.
(3) remove through genome Southern trace diagnosis drug resistant gene
According to following steps, the drug resistant gene removal of the positive colony (4-5-C3) of acquisition in (2) item is diagnosed through genome Southern trace.
In (2) between the motherboard of freezing state storage, it is dull and stereotyped and at 5%CO to select to contain 96 holes of above positive colony 2Placed 10 minutes for following 37 ℃.After the placement, be inoculated on the 24 hole flat undersides (Greiner production) that attach cell corresponding to the cell in above clone hole.After in the IMDM substratum (Invitrogen production) that has added 10% foetal calf serum (Invitrogen production) and 1x concentration HT additive (Invitrogen production), cultivating for 1 week, cell is seeded in the 6 hole flat undersides (Greiner production) that are used to attach cell.[Nucleic Acids Research in accordance with known methods; 3; 2303 (1976)] each cloned genes group DNA of preparation from flat board; And dissolving is spent the night in 150 μ l TE-rnase buffer liquid (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA, 200 μ g/ml ribonuclease As).
Contain the genomic dna that dissolves 12 μ g above-prepared in 100 μ g/ml BSA (New England Biolabs production) the NE damping fluids 2 (New England Biolabs production) at 120 μ l; To wherein adding 20 unit limit property enzyme NheI (New England Biolabs production), 37 ℃ of digestion are subsequently spent the night.From reaction soln, collect dna fragmentation through ethanol precipitation, be dissolved in the 20 μ l TE damping fluids (pH 8.0) (10mmol/l Tris-HCl, 1mmol/l EDTA), and carry out 0.6% (w/v) agarose gel electrophoresis.Behind the electrophoresis, genomic dna through known method [Proc.Natl.Acad.Sci.USA, 76, 3683 (1979)] transfer on the nylon membrane.After the transfer, nylon membrane was 80 ℃ of heating 2 hours.
Respectively, the probe preparation that is used for the Southern trace as follows.At first, the sequence bonded primer (SEQ ID NO:10 and 11) of part outside the target carrier homologous region carries out PCR according to following steps in use and the FUT8 genome district.Specifically; Prepare 20 μ l reaction solns [archaeal dna polymerase ExTaq (Takara Shuzo production); ExTaq damping fluid (Takara Shuzo production); 0.2mmol/l dNTPs, the gene specific primer (SEQ ID NOs:11 and 12) that 0.5 μ mol/l is above-mentioned], said reaction soln contains the plasmid pFUT8fgE2-2 that obtains in the 4.0ng reference implementation example (2); Through 94 ℃ of heating 1 minute, 94 ℃ 30 minutes, 55 ℃ of 25 round-robin 30 minutes and 74 ℃ of reactions in 1 minute were subsequently carried out PCR as a circulation.Behind the PCR, reaction soln carries out 1.75% (w/v) agarose gel electrophoresis, and approximately the probe dna fragment of 230bp is by purifying.Adopt [α- 32P] dCTP1.75MBq and Megaprime dna marker system, dCTP (Amersham Pharmacia Biotech production) carry out radio-labeled to 5 μ l dna probe solution.The outer sequence bonded primer (SEQ ED NOs:9 and 10) of target carrier homologous region in the FUT8 genome district that obtains in design and the reference implementation scheme.
Hybridization is carried out as follows.At first, above-mentioned nylon membrane is encapsulated in and rolls in the bottle, adds 15ml hybridization solution [5 * SSPE, 50 * Denhalt ' s solution, 0.5% (w/v) SDS, 100 μ g/ml salmon sperm DNAs] and hybridizes in 3 hours at 65 ℃.Then, use 32The dna probe of P-mark is by thermally denature and pour in the bottle, and 65 ℃ of heated overnight.
After the hybridization, in 50ml 2 * SSC-0.1% (w/v) SDS, soak nylon membrane and 65 ℃ the heating 15 minutes.After repeating twice of above-mentioned flushing operation, in 50ml 0.2 * SSC-0.1% (w/v) SDS, soak film and 65 ℃ of heating 15 minutes.After the flushing, nylon membrane contacts to develop with X line film at-80 ℃.
Handle through above-mentioned Restriction Enzyme NheI, from wild-type FUT8 allelotrope, formed the dna fragmentation of about 8.0Kb.In addition, handle, from the allelotrope that the targeting vector homologous recombination has taken place, obtained the dna fragmentation of about 9.5Kb through identical Restriction Enzyme.And, when neomycin resistance gene (approximately 1.6Kb) or puromycin resistance gene (approximately 1.5kb) when from the allelotrope that homologous recombination has taken place, being removed, have obtained the dna fragmentation of about 8.0Kb with identical processing.
Through present method, from the genomic dna of the positive colony 4-5-C3 that detects (embodiment 4 (3) positive colony), only found the homologous recombination district specific fragment (Figure 18) of about 8.0Kb.
Embodiment 6
The expression of antibody molecule in the two CHO/DG44 cells that knock out of FUT8 allelotrope:
1. prepare the CD 20 antagonizing Chimeric antibody expression vector
(1) cDNA of the anti-CD20 mouse monoclonal antibody VL of structure coding
The following PCR of employing makes up the cDNA (SEQ ID NO:17) of the aminoacid sequence of the anti-CD20 mouse monoclonal antibody 2B8VL of coding, and said antibody is described in WO94/11026.
At first, the combination nucleotide sequences of pcr amplification primer (being included as the Restriction Enzyme recognition site of humanized antibody cloning by expression to the carrier) be added to 5 of the VL nucleotide sequences described among the WO94/11026 '-terminal and 3 '-end.The nucleotide sequences of design from 5 '-end side is divided into 6 nucleotide sequences altogether; Each about 100 base (mode that has the terminal common sequences of about 20 bases with its afterbody is regulated adjacent nucleotide sequences), and with 6 synthetic DNA of the alternating sequence of positive-sense strand and antisense strand preparation, SEQ ID NOs:19; 20; 21,22,23 and 24 (entrusting GENSET company).
Each oligonucleoside adds 50 μ L reaction solns [KOD archaeal dna polymerase affixture PCR damping fluid #1 (Toyobo production); 0.2mM dNTPs; The 1mM magnesium chloride; 0.5 μ M M13 primer M4 (Takara Shuzo production), 0.5 μ M M13 primer RV (Takara Shuzo production)] in to final concentration 0.1 μ M.Adopt DNA thermal cycler GeneAmp PCR System 9600 (Perkin Elmer productions); After adding 2.5 unit K OD archaeal dna polymerases (Toyobo production); 94 ℃ of reacting by heating solution 3 minutes; 94 ℃ 30 seconds, 55 ℃ of 25 round-robin 30 seconds and ℃ 1 minute be heated to be a circulation subsequently are further 72 ℃ of heating 10 minutes.Then, 25 μ L reaction solns carry out agarose gel electrophoresis to collect the PCR product of about 0.44kb VL with QIAquick Gel Extraction Kit (QIAGEN production).
Then; The DNA 0.1 μ g that from plasmid pBluescriptllSK (-) (Stratagene production), obtains through Restriction Enzyme SmaI (Takara Shuzo production) and above add sterilized water to TV 7.5 μ L in about 0.1 μ g PCR product of acquisition; To the solution I and the 0.3 μ L Restriction Enzyme SmaI (Takara Shuzo production) that wherein add 7.5 μ L TAKARA connection test kit ver.2 (Takara Shuzo production), subsequently 22 ℃ of reactions 2 hours.Adopt the plasmid recombinant dna solution transformed into escherichia coli DH5 α strain (Toyobo production) that so obtains.Each DNA of preparation from the transformant clone; React according to appended shop instruction with BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (Applied Biosystems production), the dna sequencing appearance ABI PRISM 377 with same company analyzes nucleotide sequences then.Thereby, obtain the plasmid pBS-2B8L that shows among Figure 19 with purpose nucleotide sequences.
(2) cDNA of the anti-CD20 mouse monoclonal antibody VH of structure coding
The following PCR of employing makes up the cDNA (SEQ ID NO:18) of the aminoacid sequence of the anti-CD20 mouse monoclonal antibody 2B8 VH of coding, and said antibody is described in WO94/11026.
At first, the combination nucleotide sequences of pcr amplification primer (being included as the Restriction Enzyme recognition sequence of humanized antibody cloning by expression to the carrier) be added to 5 of the VH nucleotide sequences described among the WO94/11026 '-terminal and 3 '-end.The nucleotide sequences of design from 5 '-end side is divided into 6 nucleotide sequences altogether; Each about 100 base (mode that has the terminal common sequences of about 20 bases with its afterbody is regulated adjacent nucleotide sequences), and with 6 synthetic DNA of the alternating sequence of positive-sense strand and antisense strand preparation, SEQ ID NOs:25; 26; 27,28,29 and 30 (entrusting GENSET company).
Each oligonucleoside adds 50 μ L reaction solns [KOD archaeal dna polymerase affixture PCR damping fluid #1 (Toyobo production); 0.2mM dNTPs, 1mM magnesium chloride, 0.5 μ M M13 primer M4 (Takara Shuzo production); 0.5 μ M M13 primer RV (Takara Shuzo production)] in to final concentration 0.1 μ M; With adopt DNA thermal cycler GeneAmp PCR System 9600 (Perkin Elmer production), add 2.5 unit K OD archaeal dna polymerases (Toyobo production) therein after, 94 ℃ were heated 3 minutes; 94 ℃ 30 seconds, 55 ℃ of 25 round-robin 30 seconds and ℃ 1 minute be heated to be a circulation subsequently are further 72 ℃ of heating 19 minutes.Then, 25 μ L reaction solns carry out agarose gel electrophoresis to collect the PCR product of about 0.49kb VH with QIAquick Gel Extraction Kit (QIAGEN production).
Then; Add sterilized water to TV 7.5 μ L in the DNA 0.1 μ g that from plasmid pBluescriptIISK (-) (Stratagene production), obtains through Restriction Enzyme SmaI (Takara Shuzo production) and the about 0.1 μ gPCR product of above-mentioned acquisition; To the solution I and the 0.3 μ L Restriction Enzyme SmaI (Takara Shuzo production) that wherein add 7.5 μ L TAKARA connection test kit ver.2 (Takara Shuzo Co production), 22 ℃ of reactions are subsequently spent the night.
Adopt the plasmid recombinant dna solution transformed into escherichia coli DH5 α strain (Toyobo production) that so obtains.Each DNA of preparation from the transformant clone; React according to appended shop instruction with BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (Applied Biosystems production), the dna sequencing appearance ABI PRISM 377 with same company analyzes nucleotide sequences then.Thereby, obtain the plasmid pBS-2B8H that shows among Figure 20 with purpose nucleotide sequences.
Then, the synthetic DNA that design is expressed as SEQ ID NO:31 is substituted by Pro with the amino-acid residue with position 14 from Ala, and this replacement uses the external mutant primer of LA PCR of pBluescriptII that (Takara Shuzo production) is carried out as follows through PCR.Prepare reaction soln [the LA PCR damping fluid II (producing) that 50 μ L contain the above-mentioned plasmid pBS-2B8H of 1ng by Takara Shuzo; 2.5 the TAKARA LA Taq of unit; 0.4mM dNTPs, 2.5mM magnesium chloride, 50nM T3 BcaBEST sequencing primer (Takara Shuzo production); The above-mentioned primer of 50nM is with induced mutation (SEQ ID NO:31; Produce by GENSET)] after, with DNA thermal cycler GeneAmp PCR System9600 (Perkin Elmer productions), making reaction soln carry out 94 ℃ of 25 round-robin, to heat 30 seconds, 55 ℃ 2 minutes and 72 ℃ of one fen halfs be that a round-robin reacts.Then, 30 μ L reaction solns carry out agarose gel electrophoresis, collect the PCR product of about 0.44kb and process the aqueous solution of 30 μ L with QIAquick Gel Extraction Kit (QIAGEN production).In addition, reaction soln [the LA PCR damping fluid II (by Takara Shuzo Co., Ltd. produces) that contains the above-mentioned plasmid pBS-2B8H of 1ng with 50 μ L; 2.5 the TAKARA LA Taq of unit, 0.4mM dNTPs, 2.5mM magnesium chloride; 50nM T7BcaBEST sequencing primer (Takara Shuzo Co., Ltd. produces), 50nM MUT B1 primer (Takara Shuzo Co.; Ltd. produce)], carry out PCR in an identical manner.Then, 30 μ L reaction solns carry out agarose gel electrophoresis, collect the PCR product of about 0.63kb and process the aqueous solution of 30 μ L with QIAquick Gel Extraction Kit (QIAGEN production).Subsequently; 0.5 the PCR product of the about 0.44kb that obtains respectively above the μ L is added into 47.5 μ L reaction solns [LA PCR damping fluid II (Takara Shuzo production) with the PCR product of about 0.63kb; 0.4mM dNTPs; 2.5mM magnesium chloride] in, with DNA thermal cycler GeneAmp PCR System 9600 (Perkin Elmer production), reaction soln is reacted as follows: 90 ℃ were heated 10 minutes; Be cooled to 37 ℃ subsequently and surpass 60 minutes, thereby keep 37 ℃ of temperature to carry out DNA annealing in 15 minutes then.Add the 2.5 TAKARA LA Taq of unit (Takara Shuzo production) and 72 ℃ the reaction 3 minutes after; Add 10pmol T3 BcaBEST sequencing primer (Takara Shuzo production) and T7 BcaBEST sequencing primer (Takara Shuzo production) respectively; Reaction soln is processed 50 μ L and carried out circulating reaction 10 times: 94 ℃ of heating 30 seconds, 55 ℃ of 2 minutes and 72 ℃ of one fen halfs are a circulation.Then; With QIA fast PCR purification kit (QIAGEN production) purifying 25 μ L reaction solns; Half amount was reacted 1 hour at 37 ℃ with 10 unit limit property enzyme KpnI (Takara Shuzo Co., Ltd. produces) and 10 unit limit property enzyme SacI (Takara Shuzo Co., Ltd. produces).Through the agarose gel electrophoresis fractionation reaction soln and the KpnI-SacI fragment of collecting about 0.59kb.
Then; Reacted 1 hour with 1 μ g pBluescriptll SK (-) (Stratagene production) at 37 ℃ with 10 unit limit property enzyme KpnI (Takara Shuzo production) and 10 unit limit property enzyme SacI (Takara Shuzo production); Then, through agarose gel electrophoresis fractionation reaction soln to collect the KpnI-SacI fragment of about 2.9kb.
Adopt the solution I of DNA Ligation Kit Ver.2 (Takara Shuzo productions), connect derived from the KpnI-SacI fragment of the PCR product of acquisition as stated with derived from the KpnI-SacI fragment of plasmid pBluescriptII SK (-) according to appended shop instruction.Adopt the plasmid recombinant dna solution transformed into escherichia coli DH5 α strain (Toyobo production) that so obtains.Each DNA of preparation from the transformant clone; React according to appended shop instruction with BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (Applied Biosystems production), the dna sequencing appearance ABI PRISM 377 with same company analyzes nucleotide sequences then.
Thereby, obtain the plasmid pBS-2B8Hm that shows among Figure 20 with purpose nucleotide sequences.
(3) expression vector of the anti-CD20 people's chimeric antibody of structure.
Be adopted as the carrier pKANTEX93 that humanized antibody expresses [Mol.ImmunoL, 37, 1035 (2000)] and reach the plasmid pBS-2B8L and the pBS-2B8Hm that obtain in (1) and (2) item, the expression vector pKANTEX2B8P of anti-CD20 people's chimeric antibody (after this being called " CD 20 antagonizing Chimeric antibody ") makes up as follows.
After 1 hour, further use 10 unit limit property enzyme EcoRI (Takara Shuzo production) to react 1 hour 55 ℃ of reactions with 10 unit limit property enzyme BsiWI (New England Biolabs production) at 2 μ g (1) the plasmid pBS-2B8L that obtain at 37 ℃.Through agarose gel electrophoresis fractionation reaction soln to collect the BsiWI-EcoRI fragment of about 0.41kb.
Then; The plasmid pKANTEX93 that 2 μ g humanized antibodies are expressed reacted 1 hour at 55 ℃ with 10 unit limit property enzyme BsiWI (New England Biolabs production), and further used 10 unit limit property enzyme EcoRI (Takara Shuzo production) to react 1 hour at 37 ℃.Through agarose gel electrophoresis fractionation reaction soln to collect the BsiWI-EcoRI fragment of about 12.75kb.
Then; Adopt the solution I of DNA Ligation Kit Ver.2 (Takara Shuzo productions), acquisition derived from the BsiWI-EcoRI fragment of plasmid pBS-2B8L with derived from the BsiWI-EcoRI fragment of plasmid pKANTEX93 above connecting according to appended shop instruction.Adopt the plasmid recombinant dna solution transformed into escherichia coli DH5 α strain (Toyobo production) that so obtains to obtain the plasmid pKANTEX2B8-L that Figure 21 shows.
Then; 2 μ g (2) the plasmid pBS-2B8Hm that obtain are with 10 unit limit property enzyme ApaI (Takara Shuzo Co.; Ltd. produce) 37 ℃ of reactions 1 hour, further use 10 unit limit property enzyme NotI (Takara Shuzo Co., Ltd produces) to react 1 hour again at 37 ℃.Through agarose gel electrophoresis fractionation reaction soln to collect the ApaI-NotI fragment of about 0.45kb.
Then; 3 μ g plasmid pKANTEX2B8-L are with 10 unit limit property enzyme ApaI (Takara Shuzo Co.; Ltd. produce) 37 ℃ of reactions 1 hour, further use 10 unit limit property enzyme NotI (Takara Shuzo Co., Ltd produces) to react 1 hour again at 37 ℃.Through agarose gel electrophoresis fractionation reaction soln to collect the ApaI-NotI fragment of about 13.16kb.
Then; Adopt the solution I of DNA Ligation Kit Ver.2 (Takara Shuzo productions), acquisition derived from the ApaI-NotI fragment of plasmid pBS-2B8Hm with derived from the ApaI-NotI fragment of plasmid pKANTEX2B8-L above connecting according to appended shop instruction.Adopt the plasmid recombinant dna solution transformed into escherichia coli DH5 α strain (Toyobo production) that so obtains, from each DNA of transformant clone preparation.
With the plasmid that obtains, adopt BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (Applied Biosystems production), with the dna sequencing appearance ABI PRISM 377 analysis nucleotide sequences of same company.The result confirms, has obtained the plasmid pKANTEX2B8P with the target DNA clone that Figure 21 shows.
2. the expression of CD 20 antagonizing Chimeric antibody
The FUT8 Gene Double that the anti-CD20 antibodies expression vector pKANTEX2B8P of 1 acquisition of present embodiment is imported into 3 preparations of embodiment 5 knocks out among the clone WK704.
As follows through electroporation [Cytotechnology (electroporation), 3, 133 (1990)] and the plasmid pKANTEX2B8P gene that carries out WK704 imports.At first, 10 μ g plasmid pKANTEX2B8P are dissolved in the 100 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 40 unit limit property enzyme AatII (New England Biolabs production), linearizing was carried out in 37 ℃ of digestion in 2 hours then.With benzene/chloroform extracting abstraction reaction solution, ethanol sedimentation subsequently, and the linear plasmid that reclaims processed the aqueous solution of 1 μ g/ μ l.Respectively, WK704 is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after the above-mentioned linearization plasmid of 4 μ l (4 μ g) combines; All cell-DNA mixture is transferred to Gene Pulser Cuvette (electrode distance: 2mm) (produced by BIO-RAD), carry out gene with cytogamy equipment Gene Pulser (being produced by BIO-RAD) with 350V pulse pressure and 250 μ F electric capacity and import.After gene imports; Cell suspending liquid is suspended in the IMDM substratum (being produced by Invitrogen) that has added 10% foetal calf serum (being produced by Invitrogen) and 1 * concentration HT additive (Invitrogen production), and is seeded on the T75 flask (Greiner production) of adherent cell cultivation.At 5%CO 2In 37 ℃ cultivate after 24 hours, remove the culture supernatant thing, added the IMDM substratum (Invitrogen production) of 10% tire ox dialysis serum (Invitrogen production) to wherein adding 10ml.Whenever repeated to change substratum and cultivated 15 days, obtain the WK704-2B8P transformant at a distance from 3 to 4 days.In addition; Clone WK704-2B8P is stored in International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6 with the name of WK704-2B8P on March 20th, 2003; 1; Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566Japan), as FERM BP-8337.
3. the expression of anti-Ganglioside, GD3 chimeric antibody
The FUT8 Gene Double that the vector plasmid pKANTEX641 that expresses anti-Ganglioside, GD3 chimeric antibody is imported into preparation among 3 of embodiment 5 knocks out among the clone WK704, prepares the stably express clone of anti-GD3 chimeric antibody.PKANTEX641 is the verivate that comprises vector plasmid pChi641LHGM4 and carrier pKANTEX93, and the former expresses the anti-GD3 chimeric antibody of describing among the WOOO/61739, the latter express humanized antibody [Mol.Immunol., 37, 1035 (2000)], wherein contain from the EcoRI-HindIII fragment of the series connection antibody expression unit of pChi641LHGM4 and be connected with the EcoRI-HindIII fragment that contains from the replication initiation of pKANTEX93.
As follows through electroporation [Cytotechnology (electroporation), 3, 133 (1990)] and the plasmid pKANTEX641 gene that carries out WK704 imports.At first, 10 μ g plasmid pKANTEX641 are dissolved in the 100 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 40 unit limit property enzyme AatII (New England Biolabs production), linearizing was carried out in 37 ℃ of digestion in 2 hours then.With benzene/chloroform extracting abstraction reaction solution, ethanol sedimentation subsequently, and the linear plasmid that reclaims processed the aqueous solution of 1 μ g/ μ l.Respectively, WK704 is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with the above-mentioned linearization plasmid of 4 μ l (4 μ g) mixed after; All cell-DNA mixture is transferred to Gene Pulser Cuvette (electrode distance: 2mm) (produced by BIO-RAD), carry out gene with cytogamy equipment Gene Pulser (being produced by BIO-RAD) with 350V pulse pressure and 250 μ F electric capacity and import.After gene imports; Cell suspending liquid is suspended in the IMDM substratum (being produced by Invitrogen) that has added 10% foetal calf serum (being produced by Invitrogen) and 1 * concentration HT additive (Invitrogen production), and is seeded on the T75 flask (Greiner production) of adherent cell cultivation.At 5%CO 2In 37 ℃ cultivate after 24 hours, remove the culture supernatant thing, added the IMDM substratum (Invitrogen production) of 10% tire ox dialysis serum (Invitrogen production) to wherein adding 10ml.Whenever repeated to change substratum and cultivated 15 days, obtain transformant WK704-2871 at a distance from 3 to 4 days.In addition; Clone WK704-2871; With the name of WK704-2871 on March 20th, 2003 at International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6,1; Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan) is stored as FERM BP-8336.
4. the expression of anti-CCR4 chimeric antibody
The FUT8 Gene Double that the carrier pKANTEX2160 that expresses the anti-CCR4 chimeric antibody that WO 01/64754 describes is imported into preparation among 3 of embodiment 5 knocks out among the clone WK704, prepares the stably express clone of anti-CCR4 chimeric antibody.
Carry out as follows electroporation technology [Cytotechnology (electroporation), 3, 133 (1990)] and the gene of plasmid pKANTEX2160 is imported WK704.At first, 15 μ g plasmid pKANTEX2160 are dissolved in the 100 μ l NE damping fluids 4 (New England Biolabs production), and to wherein adding 40 unit limit property enzyme AatII (New England Biolabs production), linearizing was carried out in 37 ℃ of digestion in 2 hours then.With benzene/chloroform extracting abstraction reaction solution, ethanol sedimentation subsequently, and the linear plasmid that reclaims processed the aqueous solution of 1 μ g/ μ l.Respectively, WK704 is suspended in K-PBS damping fluid (137mmol/l KCl, 2.7mmol/l NaCl, 8.1mmol/l Na 2HPO 4, 1.5mmol/l KH 2PO 4, 4.0mmol/l MgCl 2) in to 8 * 10 7Cell/ml density.200 μ l cell suspending liquids (1.6 * 10 6Cell) with after the above-mentioned linearization plasmid of 4 μ l (4 μ g) combines; All cell-DNA mixture is transferred to Gene Pulser Cuvette (electrode distance: 2mm) (produced by BIO-RAD), carry out gene with cytogamy equipment Gene Pulser (being produced by BIO-RAD) with 350V pulse pressure and 250 μ F electric capacity and import.After gene imports; Cell suspending liquid is suspended in the IMDM substratum (being produced by Invitrogen) that has added 10% foetal calf serum (being produced by Invitrogen) and 1 * concentration HT additive (Invitrogen production), and is seeded on the T75 flask (Greiner production) of adherent cell cultivation.At 5%CO 2In 37 ℃ cultivate after 24 hours, remove the culture supernatant thing, added the IMDM substratum (Invitrogen production) of 10% tire ox dialysis serum (Invitrogen production) to wherein adding 10ml.Whenever repeated to change substratum and cultivated 15 days, obtain transformant WK704-2760 at a distance from 3 to 4 days.In addition; Clone WK704-2760; With the name of WK704-2760 on March 20th, 2003 at International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6,1; Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan) preservation is FERM BP-8335.
5. the purifying of antibody molecule
The clone WK704-2B8P of the expression anti-CD20 antibodies that in 2 of this embodiment, obtains is suspended in the IMDM substratum (Invitrogen production) that has added 10% tire ox dialysis serum (Invitrogen production) to 3 * 10 5Cell/ml density, 300ml TV are seeded on the T182 flask (Greiner production) of 10 attaching cell cultures.The clone WK704-2760 of the anti-CCR4 antibody of expression that obtains in 4 of the clone WK704-2871 of the anti-GD3 antibody of expression that obtains in 3 of this embodiments and this embodiments inoculates in an identical manner.Cultivate after 3 days, remove all culture supernatant things of each clone and change EXCELL301 substratum (JRH Biosciences production) into.They are at 5%CO 2Cultivated 7 days for 37 ℃ in the incubator, collect each cell suspending liquid then.Each of all cells suspension-s of collecting to reclaim the supernatant thing, is used PES film (Asahi Technoglass production) the filtration supernatant thing of 0.22 μ m aperture and 500ml volume with centrifugal 10 minutes of 3000rpm and 4 ℃ then.
Filling 0.5ml Mab Select on the post of diameter 0.8cm (Amersham Pharmacia Biotech production) sequentially is filled into 3.0ml purified water and 3.0ml 0.2mol/l boric acid-0.15mol/l NaCl damping fluid (pH 7.5) in the pipe then.In addition, with 2.0ml 0.1mol/l citrate buffer (pH 3.5) and 1.5ml 0.2mol/l boric acid-0.15mol/l NaCl damping fluid (pH7.5) continuous wash with the balance carrier.Then, after the above-mentioned supernatant thing of 300ml is filled on the post, with 3.0ml 0.2mol/l boric acid-0.15mol/l NaCl damping fluid (pH 7.5) flushing.After the flushing, the antibody that adsorbs on the carrier is with 1.25ml 0.1mol/l citrate buffer (pH 3.5) wash-out.After the wash-out of first 250 μ l partly is processed, reclaim next wash-out part 1ml, through being neutralized with 200 μ l2mol/l Tris-HCl (pH 8.5) are mixed.The elute soln that obtains with 10mol/l Hydrocerol A-0.15mol/l NaCl damping fluid (pH 6.0) 4 ℃ of dialysed overnight.After the dialysis, reclaim antibody-solutions, sterilization and with Millex GV (MILLIPORE production) filtration in 0.22 μ m aperture.
Embodiment 7
Cell in vitro cytotoxic activity (ADCC is active) by the FUT8 allelotrope pair antibody compositions that the CHO/DG44 cells that knock out produce:
For the cell in vitro cytotoxic activity of the anti-CD20 antibodies of estimating purifying in the embodiment 6, ADCC is active to be detected as follows.
(1) preparation target cell suspension liquid
The human B lymphocyte culturing cell of cultivating in RPMI1640-FCS (10) substratum [having added the PRMI1640 substratum (being produced by GIBCO BRL) of 10%PCS] through the spinning and the flushing that suspends with RPMI1640-FCS (5) substratum [having added the PRMI1640 substratum (being produced by GIBCO BRL) of 5%PCS] is Raji cell (JCRB9012).Then, with RPMI1640-FCS (5) medium preparation suspension-s to 2 * 10 6Cell/ml density is as target cell suspension liquid.
(2) preparation effector cell suspension-s
After gathering 50ml healthy individuals venous blood, add 0.5ml heparin sodium (producing) therein, mix lightly subsequently by Shimizu Seiyaku.With Lymphoprep (producing) by AXIS SHIELD according to the working instructions centrifugal mixture (800g, 20 minutes) of producer with the separating monocytic cell phase.With RPMI1640-FCS (5) substratum through spinning cells washed 3 times, and with identical substratum resuspension to 4 * 10 6Cell/ml density, the suspension-s that obtains is used as effector cell's suspension-s.
(3) the active detection of ADCC
Distribution 50 μ l (1 * 10 in each hole of 96 hole U-shape base plates (producing) by Falcon 4Cells/well) in the above the target cell suspension liquid for preparing in (1) item.Then, add effector cell's suspension-s 50 μ l (2 * 10 of preparation in top (2) item 5Cells/well, effector cell and target cell ratio become 20: 1).In addition, add various CD 20 antagonizing Chimeric antibodies to final concentration and be 0.3, reacted 4 hours at 37 ℃ to 3000ng/ml and TV 150 μ l.After the reaction, centrifugal flat board also passes through CytoTox96 non-radioactive active cells toxicity test (being produced by Promega) and detects serum lactic dehydrogenase (LDH) activity in the supernatant thing.Calculate the LDH amount of the spontaneous release of target cell through carrying out above-mentioned identical step, do not have effector cell's suspension-s and the antibody-solutions except that only using substratum, and the LDH that measures in the supernatant thing is active.Obtain the absorbance data of the spontaneous release of effector cell through carrying out above-mentioned identical step, do not have effector cell's suspension-s and the antibody-solutions except that only using substratum.Measure the active calculating of the LDH total free LDH amount downright bad relevant in the supernatant thing through carrying out above-mentioned identical step with all target cells; Do not have effector cell's suspension-s and the antibody-solutions except that only using substratum, added 15 μ l 9%Triton X-100 solution in preceding 45 minutes in the stopping of reaction.It is active to calculate ADCC according to following formula (II) with these values.
The ADCC activity of each anti-CD20 antibodies is presented among Figure 22.The antibody that knocks out clone WK704-2B8P acquisition from the FUT8 Gene Double is at the commercial Rituxan that obtains of all ACs ratios TMShow that higher ADCC is active, and maximum cell cytotoxic activity value is also higher.Rituxan TMBe the CD 20 antagonizing Chimeric antibody that produces as host cell with Chinese hamster ovary celI, wherein the FUT8 gene is less than fracture.In addition; Knocking out clone WK704-2871 from the FUT8 Gene Double shows with the active detected result of each antibody A DCC that clone WK704-2760 obtains; With regard to anti-CD20 antibodies, having obtained than the common Chinese hamster ovary celI that FUT8 gene does not rupture in the same manner is the higher cytotoxic activity of antibody that produces.Find according to above result,, adopt the host cell of FUT8 allelotrope fracture can prepare antibody with higher cytotoxic activity with adopting the FUT8 gene not have the host cell of fracture to compare.
Embodiment 8
The sugar chain analysis of the antibody compositions that in the two CHO/DG44 cells that knock out of FUT8 allelotrope, produces:
According to the method for describing in 2 (4) of the embodiments, the FUT8 Gene Double of carrying out in embodiment 6, obtaining knocks out the sugar chain analysis of anti-CD20 antibodies, anti-GD3 antibody and anti-CCR4 antibody that the clone produces.And, according to known method [Journal of Liquid Chromatography, 6, 1577, (1983)] and the sugar chain compsn of antagonist carries out the analysis of monose compsn.As a result, any antibody that knocks out clone WK704 acquisition from the FUT8 Gene Double, there is not to find to contain the sugar chain structure of Fucose.Show that according to The above results through at host cell cleaved FUT8 allelotrope, 1 that can fully remove Fucose in the sugar chain that compound N-glucosides connects through 6 the bonded functions of α-key at reducing end and N-acetylglucosamine.
The reference implementation example
Preparation Chinese hamster ovary celI FUT8 gene:
(1) preparation Chinese hamster ovary celI FUT8cDNA sequence
From 8 (1) of WO 00/61739 embodiments, cultivate the strand cDNA of the 2nd day CHO/DG44 cell preparation, obtain Chinese hamster FUT8cDNA (Figure 23) through following steps.
At first, (GenBank, AB025198) design is held the special forward primer (being shown as SEQ ID NO:7) of non-translational region and is held the special reverse primer (being shown as SEQ ID NO:8) of non-translational region to 3 ' 5 ' from mouse FUT8cDNA sequence.
Then; Preparation contains 25 μ l reaction solns [the ExTaq damping fluid (being produced by Takara Shuzo) of 1 μ l CHO/DG44 cell source cDNA; 0.2mmol/l dNTPs; 4%DMSO and 0.5 μ mol/l special primer (SEQ ID NOs:7 and 8)], adopt archaeal dna polymerase ExTaq (producing) to carry out PCR by Takara Shuzo.The PCR reaction conditions: 94 ℃ of heating 1 minute, 30 circulations of 94 ℃ of reactions in 30 seconds subsequently, 55 ℃ 30 seconds with 72 ℃ of circulations in 2 minutes, heated 10 minutes at 72 ℃ again.
Behind the PCR, reaction soln carries out 0.8% agarose gel electrophoresis, and about 2Kb fragment of specific amplified is by purifying.According to the appended working instructions of TOPO TA clone test kit (producing) 4 μ l dna fragmentations are inserted among the plasmid pCR2.1, with reaction soln transformed into escherichia coli DH5 α strain by Invitrogen.In accordance with known methods in the kantlex resistance clone who obtains from 8 clones that insert cDNA isolated plasmid dna.
Adopt dna sequencing appearance 377 (producing) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (producing), measure the cDNA nucleotide sequence that each inserts plasmid according to the working instructions of producer by Parkin Elmer by Parkin Elmer.This method confirms that the cDNA coding of all insertions contains the sequence of the full ORF of Chinese hamster ovary celI FUT8.Therein, select to pass through in the sequence DNA of the complete alkali-free base of PCR reading mistake.At this, plasmid is called CHfFUT8-pCR2.1.The nucleotide sequence of the CHO FUT8cDNA that measures and aminoacid sequence are shown by SEQ ID NOs:1 and 4 respectively.
(2) preparation Chinese hamster ovary celI FUT8 genome sequence
The Chinese hamster ovary celI FUT8ORF full-length cDNA fragment that obtains in the employing project (1) is as probe, according to like molecular cloning, and second edition; The molecular biology universal method; Known group screening method described in the laboratory manual, second edition (1989) obtains Chinese hamster ovary celI FUT8 genomic clone.Then; Behind the genomic clone with various Restriction Enzyme digestion acquisitions; (approximately 280bp) carries out Southern hybridization as probe with the AfaI-Sau3AI fragment that contains Chinese hamster ovary celI FUT8cDNA initiator codon; From the Restriction Enzyme fragment that shows positive reaction, select XbaI-XbaI fragment (approximately 2.5Kb) and SacI-SacI fragment (approximately 6.5Kb) then, insert respectively among the pBluescript II KS (+) (producing) by Stratagene.
Adopt dna sequencing appearance 377 (producing) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (producing), measure the nucleotide sequence of the genomic fragment of each acquisition according to the working instructions of producer by Parkin Elmer by Parkin Elmer.Therefore confirm that the XbaI-XbaI segment encoding contains the upper reaches intron sequences of about 2.5Kb of Chinese hamster ovary celI FUT8 exon 2, the SacI-SacI segment encoding contains the downstream intron sequences of about 6.5Kb of Chinese hamster ovary celI FUT8 exon 2.At this, contain the segmental plasmid of XbaI-XbaI and be called pFUT8fgE2-2, contain the segmental plasmid of SacI-SacI and be called pFUT8fgE2-4.The genome area nucleotide sequence of being measured that contains Chinese hamster ovary celI FUT8 exon 2 (approximately 9.0Kb) is shown by SEQ ID NO:3.
Industrial applicibility
The invention provides the cell that genome is modified; Said modification makes and more reduces than its parental cell with sugar chain modified involved enzyme activity or disappear; In said modification; 1 of Fucose with the N-acetylglucosamine 6 are connected through α-key at reducing end in the sugar chain that compound N-glucosides connects; Provide and used this cell and, antibody compositions of producing through said working method and the medicine that contains said antibody compositions are provided by the method for the transgenic nonhuman animal or the plant production antibody of this cell preparation.
Text in the sequence list
SEQ ID NO:7-explanation synthetic sequence: synthetic DNA
SEQ ID NO:8-explanation synthetic sequence: synthetic DNA
SEQ ID NO:9-explanation synthetic sequence: synthetic DNA
SEQ ID NO:10-explanation synthetic sequence: synthetic DNA
SEQ ED NO:11-explanation synthetic sequence: synthetic DNA
SEQ ID NO:12-explanation synthetic sequence: synthetic DNA
SEQ ID NO:13-explanation synthetic sequence: synthetic DNA
SEQ ID NO:14-explanation synthetic sequence: synthetic DNA
SEQ ID NO:15-explanation synthetic sequence: synthetic DNA
SEQ ID NO:16-explanation synthetic sequence: synthetic DNA
SEQ ID NO:19-explanation synthetic sequence: synthetic DNA
SEQ ID NO:20-explanation synthetic sequence: synthetic DNA
SEQ ED NO:21-explanation synthetic sequence: synthetic DNA
SEQ ED NO:22-explanation synthetic sequence: synthetic DNA
SEQ ID NO:23-explanation synthetic sequence: synthetic DNA
SEQ ED NO:24-explanation synthetic sequence: synthetic DNA
SEQ ID NO:25-explanation synthetic sequence: synthetic DNA
SEQ ID NO:26-explanation synthetic sequence: synthetic DNA
SEQ ED NO:27-explanation synthetic sequence: synthetic DNA
SEQ ID NO:28-explanation synthetic sequence: synthetic DNA
SEQ ED NO:29-explanation synthetic sequence: synthetic DNA
SEQ ID NO:30-explanation synthetic sequence: synthetic DNA
SEQ ID NO:31-explanation synthetic sequence: synthetic DNA
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Figure GSB00000760248001131
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Figure GSB00000760248001141
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Figure GSB00000760248001181
Pattern PCT/RO/134 (in July, 1992)
Applicant or procuratorial file number: P044080 International application no: PCT/JP03/04507
Microbial preservation proves
[PCT rule 13 2]
Figure GSB00000760248001191
Pattern PCT/RO/134 (in July, 1992)
Figure IYZ000004141214100011
Figure IYZ000004141214100051
Figure IYZ000004141214100061
Figure IYZ000004141214100091
Figure IYZ000004141214100111
Figure IYZ000004141214100121
Figure IYZ000004141214100131
Figure IYZ000004141214100151

Claims (11)

1. a modifying factor group is so that α 1; Lower or the Chinese hamster ovary celI that derives from the Chinese hamster ovary tissue that disappears of its parental cell of the specific activity of 6-fucosyltransferase; It is characterized in that described α 1, the 6-fucosyltransferase is to be selected from following (a) protein to (b):
(a) the coded protein of forming by nucleotide sequence shown in the SEQ ID NO:1 of DNA;
(b) protein of forming by aminoacid sequence shown in the SEQ ID NO:4; Wherein coding for alpha 1, and all allelotrope on the genome of 6-fucosyltransferase are knocked out.
2. cell according to claim 1; It is characterized in that said cell has resistance to the lectin of identification sugar chain, in the said sugar chain 1 of Fucose with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end pass through α-key connection.
3. cell according to claim 2 is characterized in that at least one lectin of said cell following to being selected from (a) to (d) has resistance:
(a) LcA;
(b) pisum sativum agglutinin;
(c) broad bean lectin;
(d) dried orange peel auricularia auriculajudae lectin.
4. cell according to claim 1 is characterized in that said cell contains the gene of encoding antibody molecule.
5. cell according to claim 4 is characterized in that described antibody molecule is selected from following (a) to (d):
(a) people's antibody;
(b) humanized antibody;
(c) contain (a) or (b) antibody fragment in Fc district;
(d) contain (a) or (b) fused protein in Fc district.
6. cell according to claim 4 is characterized in that described antibody molecule belongs to the IgG type.
7. cell according to claim 1 is characterized in that the antibody compositions that said cell produces has higher antibody dependent cellular mediated cell cytotoxic activity than the antibody compositions that its parental cell produces.
8. cell according to claim 7; It is characterized in that described antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity has higher sugar chain ratio than the antibody compositions that its parental cell produces, in the said sugar chain Fucose not with the total complex body of sugar chain that Fc district bonded N-glucosides is connected in the N-acetylglucosamine connection of reducing end.
9. cell according to claim 8 is characterized in that in the described sugar chain that does not connect Fucose, 1 of Fucose not with sugar chain complex body that the N-glucosides is connected in 6 of N-acetylglucosamine of reducing end connect through α-key.
10. cell according to claim 7, it is characterized in that described Fucose not with sugar chain ratio that the N-acetylglucosamine of reducing end is connected through α-key be connected with Fc district bonded N-glucosides in the antibody compositions the total complex body of sugar chain 20% or more than.
11. cell according to claim 7 is characterized in that said antibody compositions with higher antibody dependent cellular mediated cell cytotoxic activity does not have the N-acetylglucosamine bonded sugar chain of Fucose and sugar chain reducing end.
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